Friday, January 23, 2009
Lethal Factor = MMP = Metaloproteinase
Well there is absolutely no difference in MMPs that are pathologic and Lethal Factor of ANthrax fame. This 3D is of LF and where there is a will to eliminate suffering and dying from cancer and autoimmunue diseases there is a way.
Now that we know we can can busy at trying to back track from it as a PRION or other nuclearmaterial and diminish its half life by diminishing the HuR substance adherence. This we know.
Doxycycline can also act to diminish the 1/2 life of the mRNA of the MMP
Thursday, January 15, 2009
MMP
The MMP pictured should say a thousand words. After offering this for free, anyone who is not a patient will have to pay
We used to study normal histology (study of tissues) and anatomy (study of structures within and without the body) in order to be able to recognize the abnormal. Well what Peters' Productions did was propose the normal Cytokine Cycle as well as offer a software to prove it up, so that pathology in immunology can be explained in context to the normal state.
We have since been providing more data to our artificially intelligent database called AMIE (advanced medical informatic education) to relate ALL DISEASE TO IMMUNOLOGY with the CYTOKINE CYCLE.
We are not funded , yet my best effort has been put forth to allow you the patient to have access to the data. This is what we hope for. It will be from the new data and cures that scholarships and rewards will come to the kids and their kids kids. ARE WE CLEAR?
He who began a good work in you shall be faithful to complete it!
We used to study normal histology (study of tissues) and anatomy (study of structures within and without the body) in order to be able to recognize the abnormal. Well what Peters' Productions did was propose the normal Cytokine Cycle as well as offer a software to prove it up, so that pathology in immunology can be explained in context to the normal state.
We have since been providing more data to our artificially intelligent database called AMIE (advanced medical informatic education) to relate ALL DISEASE TO IMMUNOLOGY with the CYTOKINE CYCLE.
We are not funded , yet my best effort has been put forth to allow you the patient to have access to the data. This is what we hope for. It will be from the new data and cures that scholarships and rewards will come to the kids and their kids kids. ARE WE CLEAR?
He who began a good work in you shall be faithful to complete it!
Wednesday, January 14, 2009
Osteosarcoma Still no Joke 30 years later
Years ago before I received my MD I had a 14 year old patient that had a profound effect on me. He had a disease that we could not cure. I recall speaking to the family about the possibility of recurrence of the osteosarcoma and it was particularly difficult because I had gone to high school with one of the brothers of the patient. In surgery we actually saw the tumor. Unfortunately I had to help give the bad news to the family. It had a very poor prognosis.
Fortunately at the North Medical Center we did not have a patient with this type of disease but we do have more insight as to what the pathology was
The following will be posted in his memory against this dreadful disease that killed him
In hopes of helping some FP out there that has a patient with it or for family members
who would like to know more about MMPs out of control. Let us just call it dedicated to
the Cusamo family which is an abbreviation of the actual name. The AMIE post will probably not make sense but email me at pete4doc@hotmail.com for information
Fortunately at the North Medical Center we did not have a patient with this type of disease but we do have more insight as to what the pathology was
The following will be posted in his memory against this dreadful disease that killed him
In hopes of helping some FP out there that has a patient with it or for family members
who would like to know more about MMPs out of control. Let us just call it dedicated to
the Cusamo family which is an abbreviation of the actual name. The AMIE post will probably not make sense but email me at pete4doc@hotmail.com for information
or go to facebook.com
OSTEOSARCOMA/mmp
Am J Physiol Endocrinol Metab 277: E496-E504, 1999;
Am J Physiol Endocrinol Metab 277: E496-E504, 1999;
EPILOGUETriiodothyronine induces collagenase-3 and gelatinase B expression in murine osteoblasts
Renata C. Pereira1,3, Vanda Jorgetti3, and Ernesto Canalis1,2
1 Departments of Research and Medicine, Saint Francis Hospital and Medical Center, Hartford 06105; 2 University of Connecticut School of Medicine, Farmington, Connecticut 06030; and 3 Laboratorio de Fisiopatologia Renal, Universidade de Sao Paulo, Sao Paulo, Brazil 01246-903
Initiator
Prod / Activ
Prod / Activ
Jnl / Vol / Pg
Author / Yr
Misc / Vol / Ed
THYROID HORMONES
oSTEOBLASTIC PRODUCTION OF/ mmp/COLLAGENASES
bone MATRIX/EXOSKELETON
Am J Physiol Endocrinol Metab/ 277/E496-E504
Renata C. Pereira/Vanda Jorgetti/Ernesto Canalis/99
Selected effects of thyroid hormones on bone metabolism may be mediated by the production of local factors, and triiodothyronine (T3) enhances the synthesis of prostaglandin E2 and of insulin-like growth factor (IGF) I by skeletal cells (31, 55).
ABSTRACT
Triiodothyronine (T3) increases bone resorption, but its effects on matrix metalloprotease (MMP) expression in bone are unknown. We tested the effects of T3 on collagenase-3 and gelatinase A and B expression in MC3T3 osteoblastic cells. T3 at 1 nM to 1 µM for 24-72 h increased collagenase-3 and gelatinase B mRNA levels, but it did not increase gelatinase A transcripts. In addition, T3 increased immunoreactive collagenase and gelatinase activity. Cycloheximide prevented the stimulatory effect of T3 on collagenase-3 but not on gelatinase B transcripts. Indomethacin did not prevent the effect of T3 on either MMP. T3 did not alter the decay of collagenase-3 or gelatinase B mRNA in transcriptionally arrested MC3T3 cells, and it increased the rate of collagenase-3 and gelatinase B gene transcription. Although T3 enhanced the expression of the tissue inhibitor of metalloproteinase-1 in MC3T3 cells, it increased collagen degradation in cultured intact rat calvariae. In conclusion, T3 increases collagenase-3 and gelatinase B synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the actions of T3 on bone matrix remodeling.
bone remodeling; thyroid hormone; metalloprotease; collagen degradation
INTRODUCTION
THYROID HORMONES have important effects on bone metabolism and enhance bone remodeling. Thyroid hormones increase bone resorption, although their major target cell in bone probably is a cell of the osteoblastic lineage (6, 45, 59, 65). In fact, the presence of osteoblasts is required to detect an increase in bone resorption (6). Thyroid hormones also increase the replication of cells of the osteoblastic lineage and suppress the differentiation of osteoprogenitor cells to osteoblasts (13, 29, 47). Selected effects of thyroid hormones on bone metabolism may be mediated by the production of local factors, and triiodothyronine (T3) enhances the synthesis of prostaglandin E2 and of insulin-like growth factor (IGF) I by skeletal cells (31, 55).
The administration of exogenous T3 to humans results in increased bone remodeling, and postmenopausal females exposed to thyroid hormone excess display decreased bone mineral density (BMD) of the spine and hip as well as of cortical bone (1, 25, 37, 61). Some studies have failed to demonstrate marked changes in BMD, and there appears to be a relationship between the dose of thyroid hormone administered and changes in BMD (4, 22, 23, 39, 61). In addition to changes in BMD, patients with hyperthyroidism have increased urinary excretion of pyridinoline cross-links, confirming the stimulatory actions of thyroid hormone on bone remodeling and suggesting an effect on collagen matrix breakdown (21, 24). This may cause a decrease in a collagen matrix available for mineralization, and in conjunction with an increase in bone resorption, it might explain the decrease in BMD observed in these patients. Although it is tempting to believe that the major actions of thyroid hormone are on bone resorption, its direct effects on bone matrix degradation have not been explored. Bone resorption is a process mediated by the osteoclast, whereas bone matrix degradation is a process mediated by the secretion of proteases by the osteoblast. However, the two processes are closely regulated, and agents that modify bone resorption frequently alter bone collagen degradation by changing the expression of matrix metalloproteinases (MMP) and their inhibitors (9, 48). Furthermore, collagenase, an MMP, appears to play an indirect role in bone resorption (26, 27). Consequently, T3 might alter bone collagen degradation and the expression of collagenase and related metalloproteases.
MMPs are a family of related proteolytic enzymes including collagenases, gelatinases, and stromelysins (19, 41, 42). Collagenases cleave fibrillar collagen at neutral pH and are considered important in matrix remodeling. Three collagenases have been described: collagenase-1, secreted by stimulated human fibroblasts and osteoblasts and by human chondrocytes from osteoarthritic cartilage; collagenase-2, secreted by neutrophils; and collagenase-3, secreted by human breast carcinoma cells, human chondrocytes, and rodent osteoblasts (19, 43, 38, 50, 52). Unstimulated normal human osteoblasts do not secrete detectable levels of collagenase, although human osteosarcoma cell lines synthesize collagenase, and normal human osteoblasts exposed to parathyroid hormone and selected cytokines express the protease (52). Normal rat osteoblasts and rat osteosarcoma cells express collagenase-3, but rodent cells do not express collagenase-1 (48, 50). Type II collagen is preferentially hydrolyzed by collagenase-3, and collagenases-1, -2, and -3 degrade fibrillar type I collagen with similar efficiency (32). Two gelatinases have been described: gelatinase A, a 72-kDa MMP secreted by multiple cell types; and gelatinase B, a 92-kDa MMP secreted by connective tissue cells, monocytes, and various tumor cells (42). Gelatinase A and B are also expressed by skeletal cells and are known to degrade elastin, collagens IV, V, VII, X, and XI, and additional minor components of the extracellular matrix (38, 42). Agents known to enhance bone resorption increase the synthesis of gelatinases by the osteoblast (33, 38). These observations suggest a role for collagenase-3 and gelatinases in connective tissue remodeling in the skeleton, and we postulated that T3 might regulate their expression in osteoblasts.
In the present study, we examined the actions of T3 on the expression of collagenase-3, gelatinase A and B, and on tissue inhibitors of MMPs (TIMP)-1, -2, and -3 in cultures of osteoblastic MC3T3 cells and determined possible consequences in cultures of intact rat calvariae.
MATERIALS AND METHODS
Culture models. MC3T3 cells, clonal mouse osteoblastic cells derived from newborn mouse calvariae, were plated at a density of 8,000-12,000 cells/cm2 and cultured in a humidified 5% CO2 incubator at 37°C until reaching confluence (~50,000 cells/cm2; Ref. 57). Cells were cultured in -modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Summit Biotechnology, Fort Collins, CO). MC3T3 cells were grown to confluence, transferred to serum-free medium for 20 to 24 h, and exposed to test or control medium in the absence of serum for 2-72 h as indicated in the text and Figs. 1-11; 3,3',5'-triiodo-L-thyronine sodium salt (Sigma, St. Louis, MO) was added directly to the culture medium. Cycloheximide, 5,6-dichlorobenzimidazole riboside (DRB), and indomethacin (Sigma) were dissolved in ethanol and diluted 1:200 and 1:1,000, respectively, in -modified Eagle's medium. An equal amount of solvent was added to control cultures. In experiments lasting longer than 24 h, the culture medium was replaced with fresh solutions every 24 h. At the end of the incubation, the medium was harvested in the presence of 0.1% polyoxyethylenesorbitan monolaurate (Tween, Pierce, Rockford, IL) for Western blot analyses and in its absence for gelatin zymograms and then was stored at 80°C before analysis. The cell layer was extracted for RNA analysis and stored at 80°C, and nuclei were obtained by Dounce homogenization for nuclear run-on assays.
Cultures of intact rat parietal bones were used to determine changes on collagen degradation, because of the greater accumulation of collagenous protein in the matrix of intact bone than in that of monolayer cultures (54). Parietal bones were obtained from 22-day-old fetal rats immediately after the mothers were killed by blunt trauma to the nuchal area. The project was approved by the Institutional Animal Care and Use Committee of Saint Francis Hospital and Medical Center. Half-calvariae were cultured in flasks containing Biggers, Gwatkin, and Judak medium in the absence of serum. The flasks were gassed with 5% CO2, sealed and placed in a shaking water bath at 37°C for a 24-h period, and labeled with 5 µCi/ml of [2,3-3H]proline (specific activity of 40 Ci/mmol; Du Pont, Wilmington, DE). Bones were transferred to control or test medium in 10 mM proline for a 24- to 72-h "chase period." Calvariae and corresponding culture medium samples were obtained for [3H]hydroxyproline analysis (54).
Northern blot analysis. Total cellular RNA was isolated by RNeasy kit per instructions of the manufacturer (Qiagen, Chatsworth, CA). The RNA recovered was quantitated by spectrophotometry, and equal amounts of RNA from control or test samples were loaded on a formaldehyde-agarose gel after denaturation. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA, before and after transfer, documenting equal RNA loading of the samples. The RNA was blotted onto GeneScreen Plus charged nylon (Du Pont). Restriction fragments containing a 2.6-kb rat interstitial collagenase-3 cDNA (kindly provided by Cheryl Quinn, St. Louis, MO), a 1.1-kb human gelatinase A cDNA (American Type Culture Collection, Rockville, MD), a 1.4-kb murine gelatinase B cDNA (kindly provided by Ghislain Opdenakker, Leuven, Belgium), an 825-bp murine TIMP-1 cDNA, a 700-bp murine TIMP-2 cDNA, a 750-bp murine TIMP-3 cDNA (all TIMP cDNAs kindly provided by Dylan Edwards, Calgary, Alberta, Canada), and an 800-bp rat glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA (kindly provided by Ray Wu, Ithaca, NY) were labeled with [ -32P]dCTP and [ -32P]dATP (specific activity of 3,000 Ci/mmol; Du Pont) with the random hexanucleotide primed second strand synthesis method (12, 14, 35, 36, 40, 50, 60). Hybridizations were carried out at 42°C for 16-72 h. Posthybridization washes were performed in 1× saline sodium citrate (SSC) at 65°C for collagenase-3 and TIMP-1, -2, and -3 cDNAs in 0.2× SSC at 65°C for gelatinase A and B and in 0.5× SSC at 65°C for GAPD cDNA. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 or Biomax film (Eastman Kodak, Rochester, NY) or Du Pont reflection film employing intensifying screens. Relative hybridization levels were determined by densitometry. Northern analyses shown are representative of three or more cultures.
Nuclear run-on assay. To examine changes in the rate of transcription, nuclei were isolated by Dounce homogenization in a Tris buffer containing 0.5% Nonidet P-40. Nascent transcripts were labeled by incubation of nuclei in a reaction buffer containing 500 µM each of ATP, CTP, and guanidine triphosphate, 150 U of RNasin (Promega), and 250 µCi of [ -32P]uridine triphosphate (specific activity of 3,000 Ci/mmol; Du Pont; Ref. 3). RNA was isolated by treatment with DNase I and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. Linearized plasmid DNA containing ~1 µg of cDNA was immobilized onto GeneScreen Plus by slot blotting according to the directions of the manufacturer (Du Pont). The plasmid vector pUC18 (Life Technologies) was used as a control for nonspecific hybridization, and GAPD cDNA was used to estimate uniformity of radioactive counts applied to the membrane. Equal counts per minute of 32P-labeled RNA from each sample were hybridized to cDNAs with the same conditions as for Northern blot analysis and were visualized by autoradiography.
Western immunoblot analysis. Medium samples were fractionated by polyacrylamide gel electrophoresis with denaturing and nonreducing conditions and were transferred onto Immobilon P membranes (Millipore, Bedford, MA). After being blocked with 2% BSA, the membranes were exposed to a 1:1,000 dilution of rabbit antiserum raised against rat collagenase-3 (kindly provided by John Jeffrey, Albany, NY), previously characterized for specificity and immunoreactivity, followed by the addition of goat anti-rabbit IgG conjugated to horseradish peroxidase (28). The blots were washed and developed with a horseradish peroxidase chemiluminescence detection reagent (Du Pont), visualized by autoradiography on Du Pont reflection film employing reflection intensifying screens, and analyzed by densitometry. Data shown are representative of three cultures.
Gelatin zymogram. To assess gelatinase activity, aliquots of conditioned medium were mixed with sample buffer containing 2% sodium dodecyl sulfate. Samples were loaded on a 7.5% polyacrylamide gel containing 1 mg/ml of gelatin and fractionated by electrophoresis as described (33). The gels were incubated 1 h in 50 mM Tris · HCl, 5 mM CaCl2, and 1 µM ZnCl2 buffer (pH 7.5) containing 2.5% Triton X-100 (Sigma). Gels were incubated in the same buffer without Triton X-100 at 37°C overnight. Proteolytic activity was visualized by staining the gels with 0.1% Coomassie blue in 50% methanol and 20% acetic acid and destaining with 30% methanol and 1% formic acid. Data shown are representative of three cultures.
Collagen degradation assay. Calvariae were homogenized in 0.5 M acetic acid, and aliquots of the homogenate and the respective culture medium were hydrolyzed in vacuo in 6 N HCl at 107°C for 24 h. The samples were derivatized with phenylisothiocyanate, and [3H]proline and [3H]hydroxyproline were separated by reverse-phase HPLC with a C18 Nova-Pak column and an acetonitrile solvent system, as previously described (54). Although the method used to hydrolyze collagen from calvariae and medium samples may result in some destruction of hydroxyproline, this effect is minimal because it achieves nearly 100% recovery of hydroxyproline (54). [3H]hydroxyproline was measured in calvaria and medium samples by liquid scintillation counting.
Statistical methods. Data on collagenase-3 and gelatinase B mRNA and protease levels in MC3T3 cells and collagen degradation levels in calvaria are expressed as means ± SE. Statistical differences were calculated by ANOVA, and post hoc examination was performed by the Ryan-Einot-Gabriel-Welch F test (64). Data on collagenase-3 and gelatinase B mRNA decay were analyzed by linear regression, and the slopes of the regression lines obtained for control and treated cells were compared for significant differences with the method of Sokal and Rohlf (56).
Northern blot analysis of total RNA from MC3T3 cells revealed a collagenase-3 transcript of 2.9 kb (Fig. 1). Continuous treatment of MC3T3 cells with T3 at 10 nM caused a time-dependent increase in collagenase steady-state transcripts. No stimulation was observed after 2 or 6 h (not shown) of treatment with T3, whereas at 24 h collagenase-3 mRNA levels were increased by (means ± SE; n = 6) 2.8 ± 0.3-fold. The effects were maximal after 48 h and sustained for 72 h, when T3 increased collagenase transcripts by 9.8 ± 2.7- and 11.3 ± 0.8-fold, respectively (Fig. 1). The effect of T3 was dose dependent, and continued exposure of MC3T3 cells to T3 at 1-1,000 nM for 72 h increased collagenase transcripts by 3.7- to 11.3-fold (Fig. 2). T3 at 10 nM for 24, 48, or 72 h increased the levels of immunoreactive interstitial collagenase-3 in the culture medium of MC3T3 cells by (means ± SE; n = 3) 1.6 ± 0.3, 6.1 ± 1.1, and 4.5 ± 1.2, respectively (Fig. 3). Collagenase-3 was identified by comigration with a purified rat procollagenase-3 standard
The present investigation was undertaken to determine whether T3 regulates the expression of selected MMPs and TIMP-1, -2, and -3 in cultures of osteoblastic MC3T3 cells. T3 caused a time- and dose-dependent stimulation of collagenase-3 mRNA and protease levels. T3 also increased the expression of gelatinase B transcripts and activity without modifying gelatinase A mRNA levels. Although T3 increased TIMP-1 mRNA levels, the effect was modest in relation to the effect on MMPs, and T3 did not stimulate the expression of TIMP-2 or 3. Because TIMPs bind MMPs in a 1:1 stoichiometric fashion, our results suggest that the actions of the increased collagenase-3 and gelatinase B will remain unopposed by the limited expression of TIMPs in the bone microenvironment (32). This is further supported by the demonstration of an induction of active gelatinase B by T3, as detected by zymography. The stimulatory effect of T3 on collagenase and gelatinase B may be relevant to its stimulatory actions on bone resorption and probably explains the increase in bone collagen degradation observed in intact calvariae (27). However, it is important to note that this is a different model, and intact calvaraie contain not only osteoblasts but a mixed cell population and a structured matrix. Therefore, other factors in addition to the induction of collagenase-3 and gelatinase B may play a role in the collagen degradation induced by T3 in this model. The effect of T3 on collagenase transcripts was dependent on de novo protein synthesis, whereas that on gelatinase B was not, indicating that different mechanisms regulate the expression of the two MMPs by T3. Although T3 is known to induce prostaglandin E2 in osteoblasts and prostaglandins are known to enhance collagenase synthesis, we found that the stimulation of collagenase-3 and gelatinase B by T3 in MC3T3 cells was not dependent on prostaglandin synthesis (10, 31). This is in agreement with the actions of T3 on bone resorption, which can be independent of prostaglandin synthesis (31). T3 did not alter collagenase-3 or gelatinase B mRNA stability in transcriptionally arrested Ob cells but did increase the rate of transcription of the collagenase-3 and gelatinase B genes. These results indicate that T3 stimulates rat collagenase-3 and gelatinase B expression by transcriptional mechanisms. The effect of T3 on gelatinase B expression is not surprising because other agents known to induce collagenase-3 synthesis, such as interleukin-1 and -6, also increase gelatinase A or B production by skeletal cells (18, 38).
T3 induces the transcription of the collagenase-3 and gelatinase B gene in osteoblasts, but the gene sequences responsible for the effects have not been determined. In nonskeletal cells, T3 activation of other genes involves a T3 response element (TRE; Refs. 5, 30). The traditional half-site contains the sequence 5'-AGGTCA-3', although optimal binding of T3 receptors may require additional sequences and the formation of a heterodimeric and not of a homodimeric complex. Furthermore, retinoic acid receptor-binding motifs may play a role in T3-receptor binding (46). Examination of the human collagenase-3 gene revealed four TRE consensus sequences in the region of bp 1,550 to 590 (58). The sequence of the rat collagenase-3 gene is known from bp 456 to +62 and that of the murine gene is not known (51). It is possible that rodent collagenase-3 genes contain TREs in a location similar to that described for the human gene that are responsible for the transcriptional effects observed. However, the exact elements responsible for the T3 effects are not known. T3 receptors may also act by regulating activator protein-1 or -2 sequences, present in the collagenase-3 gene, as it has been reported for the activation and suppression of other genes (20, 49). Examination of the gelatinase B gene promoter reveals the presence of a TRE and activator protein-1 and -2 binding sequences, which might be responsible for the effect of T3 (53). Additional studies will be required to define the exact elements responsible for the induction of collagenase-3 and gelatinase B by T3.
The synthesis of collagenase-1 and -3 by human and rat osteoblasts is regulated by systemic hormones and by cytokines present in the bone microenvironment. Consequently, the apparent constitutive level of collagenase expression by the osteoblast is in fine balance and depends on the exposure of the cell to factors that stimulate and factors that inhibit collagenase synthesis (7, 10, 11, 17, 48, 58, 62, 63). It is possible that T3 interacts with other cytokines, such as interleukin-1 or -6, to enhance collagenase-3 expression as it has been reported for the effects of T3 on bone resorption (59). Because the effect of T3 on MMP expression is slow in onset, we tested whether or not T3 induced the expression of local factors known to stimulate collagenase expression in osteoblasts. Treatment of MC3T3 cells with T3 at 10 nM for 2-72 h did not increase the expression of platelet-derived growth factor BB, fibroblast growth factor 2, IL-1, or IL-6, all known stimulators of collagenase-3 expression (Pereira and Canalis, unpublished observations; Refs. 10, 17, 33, 62, 63). The stimulatory effect of T3 on bone resorption requires the presence of osteoblasts and depends on receptors expressed by osteoblastic cells (6, 65). Consequently, it is not surprising that T3 regulates MMP expression in osteoblasts. T3 was tested at concentrations of 1 nM and higher because previous studies from other investigators revealed that T3 was not effective in cultures of intact calvariae, long bones, and cells of the osteoblastic lineage at lower doses (31, 45, 47, 49). It is possible that under the conditions used in these experiments, concentrations lower than 1 nM could have been effective, although they were not tested. The effects of T3 on MMP expression reported here were observed at 1 nM, and this concentration is ~200-fold higher than the concentrations of free T3 found in the serum of normal humans and 50- to 100-fold higher than the concentrations found in patients with hyperthyroidism (34). The effect of T3 on collagenase and gelatinase expression reported here probably explains the increase in bone collagen degradation that we observed in rat calvariae and may be relevant to its stimulatory actions on bone resorption reported previously (6, 45, 59). It may also explain the increased bone remodeling and increased urinary excretion of collagen cross-links in vivo after thyroid hormone administration and be important in the pathogenesis of the osteopenia after thyroid hormone excess (4, 21, 24). The actions of T3 on MMP expression could have additional effects in bone metabolism because collagenase was recently implied in the fragmentation of IGF-binding protein-5, a binding protein with known stimulatory effects on bone formation (2, 14, 15, 16). Although the activity of these fragments remains to be defined, their appearance in conjunction with an increase in IGF-I might be relevant to selected anabolic actions of T3 in the skeleton (29, 55).
In conclusion, the present studies demonstrate that T3 increases collagenase-3 and gelatinase B expression in osteoblasts by transcriptional mechanisms. It is probable that these effects play a role in the degradation of the collagen matrix and in the osteopenia observed in conditions of thyroid hormone excess.
ACKNOWLEDGEMENTS
We thank Dr. Cheryl Quinn for the rat collagenase cDNA, Dr. Ghislain Opdenakker for the murine gelatinase B cDNA, Dr. Dylan Edwards for the murine TIMP-1, -2 and -3 cDNAs, Dr. Ray Wu for GAPD cDNA, and Dr. John Jeffrey for the rat interstitial collagenase antibody. We also thank Cathy Boucher, Deena Durant, and Sheila Rydziel for technical assistance and Charlene Gobeli and Margaret Nagle for secretarial help.
FOOTNOTES
This work was supported by Grant AR-21707 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. Renata C. Pereira is a recipient of a Fundaç o Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Student Fellowship Grant from the Universidade de Sao Paulo.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. Canalis, Dept. of Research, Saint Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT 06105-1299.
Received 12 February 1999; accepted in final form 29 April 1999.
REFERENCES
TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES
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Renata C. Pereira1,3, Vanda Jorgetti3, and Ernesto Canalis1,2
1 Departments of Research and Medicine, Saint Francis Hospital and Medical Center, Hartford 06105; 2 University of Connecticut School of Medicine, Farmington, Connecticut 06030; and 3 Laboratorio de Fisiopatologia Renal, Universidade de Sao Paulo, Sao Paulo, Brazil 01246-903
Initiator
Prod / Activ
Prod / Activ
Jnl / Vol / Pg
Author / Yr
Misc / Vol / Ed
THYROID HORMONES
oSTEOBLASTIC PRODUCTION OF/ mmp/COLLAGENASES
bone MATRIX/EXOSKELETON
Am J Physiol Endocrinol Metab/ 277/E496-E504
Renata C. Pereira/Vanda Jorgetti/Ernesto Canalis/99
Selected effects of thyroid hormones on bone metabolism may be mediated by the production of local factors, and triiodothyronine (T3) enhances the synthesis of prostaglandin E2 and of insulin-like growth factor (IGF) I by skeletal cells (31, 55).
ABSTRACT
Triiodothyronine (T3) increases bone resorption, but its effects on matrix metalloprotease (MMP) expression in bone are unknown. We tested the effects of T3 on collagenase-3 and gelatinase A and B expression in MC3T3 osteoblastic cells. T3 at 1 nM to 1 µM for 24-72 h increased collagenase-3 and gelatinase B mRNA levels, but it did not increase gelatinase A transcripts. In addition, T3 increased immunoreactive collagenase and gelatinase activity. Cycloheximide prevented the stimulatory effect of T3 on collagenase-3 but not on gelatinase B transcripts. Indomethacin did not prevent the effect of T3 on either MMP. T3 did not alter the decay of collagenase-3 or gelatinase B mRNA in transcriptionally arrested MC3T3 cells, and it increased the rate of collagenase-3 and gelatinase B gene transcription. Although T3 enhanced the expression of the tissue inhibitor of metalloproteinase-1 in MC3T3 cells, it increased collagen degradation in cultured intact rat calvariae. In conclusion, T3 increases collagenase-3 and gelatinase B synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the actions of T3 on bone matrix remodeling.
bone remodeling; thyroid hormone; metalloprotease; collagen degradation
INTRODUCTION
THYROID HORMONES have important effects on bone metabolism and enhance bone remodeling. Thyroid hormones increase bone resorption, although their major target cell in bone probably is a cell of the osteoblastic lineage (6, 45, 59, 65). In fact, the presence of osteoblasts is required to detect an increase in bone resorption (6). Thyroid hormones also increase the replication of cells of the osteoblastic lineage and suppress the differentiation of osteoprogenitor cells to osteoblasts (13, 29, 47). Selected effects of thyroid hormones on bone metabolism may be mediated by the production of local factors, and triiodothyronine (T3) enhances the synthesis of prostaglandin E2 and of insulin-like growth factor (IGF) I by skeletal cells (31, 55).
The administration of exogenous T3 to humans results in increased bone remodeling, and postmenopausal females exposed to thyroid hormone excess display decreased bone mineral density (BMD) of the spine and hip as well as of cortical bone (1, 25, 37, 61). Some studies have failed to demonstrate marked changes in BMD, and there appears to be a relationship between the dose of thyroid hormone administered and changes in BMD (4, 22, 23, 39, 61). In addition to changes in BMD, patients with hyperthyroidism have increased urinary excretion of pyridinoline cross-links, confirming the stimulatory actions of thyroid hormone on bone remodeling and suggesting an effect on collagen matrix breakdown (21, 24). This may cause a decrease in a collagen matrix available for mineralization, and in conjunction with an increase in bone resorption, it might explain the decrease in BMD observed in these patients. Although it is tempting to believe that the major actions of thyroid hormone are on bone resorption, its direct effects on bone matrix degradation have not been explored. Bone resorption is a process mediated by the osteoclast, whereas bone matrix degradation is a process mediated by the secretion of proteases by the osteoblast. However, the two processes are closely regulated, and agents that modify bone resorption frequently alter bone collagen degradation by changing the expression of matrix metalloproteinases (MMP) and their inhibitors (9, 48). Furthermore, collagenase, an MMP, appears to play an indirect role in bone resorption (26, 27). Consequently, T3 might alter bone collagen degradation and the expression of collagenase and related metalloproteases.
MMPs are a family of related proteolytic enzymes including collagenases, gelatinases, and stromelysins (19, 41, 42). Collagenases cleave fibrillar collagen at neutral pH and are considered important in matrix remodeling. Three collagenases have been described: collagenase-1, secreted by stimulated human fibroblasts and osteoblasts and by human chondrocytes from osteoarthritic cartilage; collagenase-2, secreted by neutrophils; and collagenase-3, secreted by human breast carcinoma cells, human chondrocytes, and rodent osteoblasts (19, 43, 38, 50, 52). Unstimulated normal human osteoblasts do not secrete detectable levels of collagenase, although human osteosarcoma cell lines synthesize collagenase, and normal human osteoblasts exposed to parathyroid hormone and selected cytokines express the protease (52). Normal rat osteoblasts and rat osteosarcoma cells express collagenase-3, but rodent cells do not express collagenase-1 (48, 50). Type II collagen is preferentially hydrolyzed by collagenase-3, and collagenases-1, -2, and -3 degrade fibrillar type I collagen with similar efficiency (32). Two gelatinases have been described: gelatinase A, a 72-kDa MMP secreted by multiple cell types; and gelatinase B, a 92-kDa MMP secreted by connective tissue cells, monocytes, and various tumor cells (42). Gelatinase A and B are also expressed by skeletal cells and are known to degrade elastin, collagens IV, V, VII, X, and XI, and additional minor components of the extracellular matrix (38, 42). Agents known to enhance bone resorption increase the synthesis of gelatinases by the osteoblast (33, 38). These observations suggest a role for collagenase-3 and gelatinases in connective tissue remodeling in the skeleton, and we postulated that T3 might regulate their expression in osteoblasts.
In the present study, we examined the actions of T3 on the expression of collagenase-3, gelatinase A and B, and on tissue inhibitors of MMPs (TIMP)-1, -2, and -3 in cultures of osteoblastic MC3T3 cells and determined possible consequences in cultures of intact rat calvariae.
MATERIALS AND METHODS
Culture models. MC3T3 cells, clonal mouse osteoblastic cells derived from newborn mouse calvariae, were plated at a density of 8,000-12,000 cells/cm2 and cultured in a humidified 5% CO2 incubator at 37°C until reaching confluence (~50,000 cells/cm2; Ref. 57). Cells were cultured in -modified Eagle's medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Summit Biotechnology, Fort Collins, CO). MC3T3 cells were grown to confluence, transferred to serum-free medium for 20 to 24 h, and exposed to test or control medium in the absence of serum for 2-72 h as indicated in the text and Figs. 1-11; 3,3',5'-triiodo-L-thyronine sodium salt (Sigma, St. Louis, MO) was added directly to the culture medium. Cycloheximide, 5,6-dichlorobenzimidazole riboside (DRB), and indomethacin (Sigma) were dissolved in ethanol and diluted 1:200 and 1:1,000, respectively, in -modified Eagle's medium. An equal amount of solvent was added to control cultures. In experiments lasting longer than 24 h, the culture medium was replaced with fresh solutions every 24 h. At the end of the incubation, the medium was harvested in the presence of 0.1% polyoxyethylenesorbitan monolaurate (Tween, Pierce, Rockford, IL) for Western blot analyses and in its absence for gelatin zymograms and then was stored at 80°C before analysis. The cell layer was extracted for RNA analysis and stored at 80°C, and nuclei were obtained by Dounce homogenization for nuclear run-on assays.
Cultures of intact rat parietal bones were used to determine changes on collagen degradation, because of the greater accumulation of collagenous protein in the matrix of intact bone than in that of monolayer cultures (54). Parietal bones were obtained from 22-day-old fetal rats immediately after the mothers were killed by blunt trauma to the nuchal area. The project was approved by the Institutional Animal Care and Use Committee of Saint Francis Hospital and Medical Center. Half-calvariae were cultured in flasks containing Biggers, Gwatkin, and Judak medium in the absence of serum. The flasks were gassed with 5% CO2, sealed and placed in a shaking water bath at 37°C for a 24-h period, and labeled with 5 µCi/ml of [2,3-3H]proline (specific activity of 40 Ci/mmol; Du Pont, Wilmington, DE). Bones were transferred to control or test medium in 10 mM proline for a 24- to 72-h "chase period." Calvariae and corresponding culture medium samples were obtained for [3H]hydroxyproline analysis (54).
Northern blot analysis. Total cellular RNA was isolated by RNeasy kit per instructions of the manufacturer (Qiagen, Chatsworth, CA). The RNA recovered was quantitated by spectrophotometry, and equal amounts of RNA from control or test samples were loaded on a formaldehyde-agarose gel after denaturation. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA, before and after transfer, documenting equal RNA loading of the samples. The RNA was blotted onto GeneScreen Plus charged nylon (Du Pont). Restriction fragments containing a 2.6-kb rat interstitial collagenase-3 cDNA (kindly provided by Cheryl Quinn, St. Louis, MO), a 1.1-kb human gelatinase A cDNA (American Type Culture Collection, Rockville, MD), a 1.4-kb murine gelatinase B cDNA (kindly provided by Ghislain Opdenakker, Leuven, Belgium), an 825-bp murine TIMP-1 cDNA, a 700-bp murine TIMP-2 cDNA, a 750-bp murine TIMP-3 cDNA (all TIMP cDNAs kindly provided by Dylan Edwards, Calgary, Alberta, Canada), and an 800-bp rat glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA (kindly provided by Ray Wu, Ithaca, NY) were labeled with [ -32P]dCTP and [ -32P]dATP (specific activity of 3,000 Ci/mmol; Du Pont) with the random hexanucleotide primed second strand synthesis method (12, 14, 35, 36, 40, 50, 60). Hybridizations were carried out at 42°C for 16-72 h. Posthybridization washes were performed in 1× saline sodium citrate (SSC) at 65°C for collagenase-3 and TIMP-1, -2, and -3 cDNAs in 0.2× SSC at 65°C for gelatinase A and B and in 0.5× SSC at 65°C for GAPD cDNA. The bound radioactive material was visualized by autoradiography on Kodak X-AR5 or Biomax film (Eastman Kodak, Rochester, NY) or Du Pont reflection film employing intensifying screens. Relative hybridization levels were determined by densitometry. Northern analyses shown are representative of three or more cultures.
Nuclear run-on assay. To examine changes in the rate of transcription, nuclei were isolated by Dounce homogenization in a Tris buffer containing 0.5% Nonidet P-40. Nascent transcripts were labeled by incubation of nuclei in a reaction buffer containing 500 µM each of ATP, CTP, and guanidine triphosphate, 150 U of RNasin (Promega), and 250 µCi of [ -32P]uridine triphosphate (specific activity of 3,000 Ci/mmol; Du Pont; Ref. 3). RNA was isolated by treatment with DNase I and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. Linearized plasmid DNA containing ~1 µg of cDNA was immobilized onto GeneScreen Plus by slot blotting according to the directions of the manufacturer (Du Pont). The plasmid vector pUC18 (Life Technologies) was used as a control for nonspecific hybridization, and GAPD cDNA was used to estimate uniformity of radioactive counts applied to the membrane. Equal counts per minute of 32P-labeled RNA from each sample were hybridized to cDNAs with the same conditions as for Northern blot analysis and were visualized by autoradiography.
Western immunoblot analysis. Medium samples were fractionated by polyacrylamide gel electrophoresis with denaturing and nonreducing conditions and were transferred onto Immobilon P membranes (Millipore, Bedford, MA). After being blocked with 2% BSA, the membranes were exposed to a 1:1,000 dilution of rabbit antiserum raised against rat collagenase-3 (kindly provided by John Jeffrey, Albany, NY), previously characterized for specificity and immunoreactivity, followed by the addition of goat anti-rabbit IgG conjugated to horseradish peroxidase (28). The blots were washed and developed with a horseradish peroxidase chemiluminescence detection reagent (Du Pont), visualized by autoradiography on Du Pont reflection film employing reflection intensifying screens, and analyzed by densitometry. Data shown are representative of three cultures.
Gelatin zymogram. To assess gelatinase activity, aliquots of conditioned medium were mixed with sample buffer containing 2% sodium dodecyl sulfate. Samples were loaded on a 7.5% polyacrylamide gel containing 1 mg/ml of gelatin and fractionated by electrophoresis as described (33). The gels were incubated 1 h in 50 mM Tris · HCl, 5 mM CaCl2, and 1 µM ZnCl2 buffer (pH 7.5) containing 2.5% Triton X-100 (Sigma). Gels were incubated in the same buffer without Triton X-100 at 37°C overnight. Proteolytic activity was visualized by staining the gels with 0.1% Coomassie blue in 50% methanol and 20% acetic acid and destaining with 30% methanol and 1% formic acid. Data shown are representative of three cultures.
Collagen degradation assay. Calvariae were homogenized in 0.5 M acetic acid, and aliquots of the homogenate and the respective culture medium were hydrolyzed in vacuo in 6 N HCl at 107°C for 24 h. The samples were derivatized with phenylisothiocyanate, and [3H]proline and [3H]hydroxyproline were separated by reverse-phase HPLC with a C18 Nova-Pak column and an acetonitrile solvent system, as previously described (54). Although the method used to hydrolyze collagen from calvariae and medium samples may result in some destruction of hydroxyproline, this effect is minimal because it achieves nearly 100% recovery of hydroxyproline (54). [3H]hydroxyproline was measured in calvaria and medium samples by liquid scintillation counting.
Statistical methods. Data on collagenase-3 and gelatinase B mRNA and protease levels in MC3T3 cells and collagen degradation levels in calvaria are expressed as means ± SE. Statistical differences were calculated by ANOVA, and post hoc examination was performed by the Ryan-Einot-Gabriel-Welch F test (64). Data on collagenase-3 and gelatinase B mRNA decay were analyzed by linear regression, and the slopes of the regression lines obtained for control and treated cells were compared for significant differences with the method of Sokal and Rohlf (56).
Northern blot analysis of total RNA from MC3T3 cells revealed a collagenase-3 transcript of 2.9 kb (Fig. 1). Continuous treatment of MC3T3 cells with T3 at 10 nM caused a time-dependent increase in collagenase steady-state transcripts. No stimulation was observed after 2 or 6 h (not shown) of treatment with T3, whereas at 24 h collagenase-3 mRNA levels were increased by (means ± SE; n = 6) 2.8 ± 0.3-fold. The effects were maximal after 48 h and sustained for 72 h, when T3 increased collagenase transcripts by 9.8 ± 2.7- and 11.3 ± 0.8-fold, respectively (Fig. 1). The effect of T3 was dose dependent, and continued exposure of MC3T3 cells to T3 at 1-1,000 nM for 72 h increased collagenase transcripts by 3.7- to 11.3-fold (Fig. 2). T3 at 10 nM for 24, 48, or 72 h increased the levels of immunoreactive interstitial collagenase-3 in the culture medium of MC3T3 cells by (means ± SE; n = 3) 1.6 ± 0.3, 6.1 ± 1.1, and 4.5 ± 1.2, respectively (Fig. 3). Collagenase-3 was identified by comigration with a purified rat procollagenase-3 standard
The present investigation was undertaken to determine whether T3 regulates the expression of selected MMPs and TIMP-1, -2, and -3 in cultures of osteoblastic MC3T3 cells. T3 caused a time- and dose-dependent stimulation of collagenase-3 mRNA and protease levels. T3 also increased the expression of gelatinase B transcripts and activity without modifying gelatinase A mRNA levels. Although T3 increased TIMP-1 mRNA levels, the effect was modest in relation to the effect on MMPs, and T3 did not stimulate the expression of TIMP-2 or 3. Because TIMPs bind MMPs in a 1:1 stoichiometric fashion, our results suggest that the actions of the increased collagenase-3 and gelatinase B will remain unopposed by the limited expression of TIMPs in the bone microenvironment (32). This is further supported by the demonstration of an induction of active gelatinase B by T3, as detected by zymography. The stimulatory effect of T3 on collagenase and gelatinase B may be relevant to its stimulatory actions on bone resorption and probably explains the increase in bone collagen degradation observed in intact calvariae (27). However, it is important to note that this is a different model, and intact calvaraie contain not only osteoblasts but a mixed cell population and a structured matrix. Therefore, other factors in addition to the induction of collagenase-3 and gelatinase B may play a role in the collagen degradation induced by T3 in this model. The effect of T3 on collagenase transcripts was dependent on de novo protein synthesis, whereas that on gelatinase B was not, indicating that different mechanisms regulate the expression of the two MMPs by T3. Although T3 is known to induce prostaglandin E2 in osteoblasts and prostaglandins are known to enhance collagenase synthesis, we found that the stimulation of collagenase-3 and gelatinase B by T3 in MC3T3 cells was not dependent on prostaglandin synthesis (10, 31). This is in agreement with the actions of T3 on bone resorption, which can be independent of prostaglandin synthesis (31). T3 did not alter collagenase-3 or gelatinase B mRNA stability in transcriptionally arrested Ob cells but did increase the rate of transcription of the collagenase-3 and gelatinase B genes. These results indicate that T3 stimulates rat collagenase-3 and gelatinase B expression by transcriptional mechanisms. The effect of T3 on gelatinase B expression is not surprising because other agents known to induce collagenase-3 synthesis, such as interleukin-1 and -6, also increase gelatinase A or B production by skeletal cells (18, 38).
T3 induces the transcription of the collagenase-3 and gelatinase B gene in osteoblasts, but the gene sequences responsible for the effects have not been determined. In nonskeletal cells, T3 activation of other genes involves a T3 response element (TRE; Refs. 5, 30). The traditional half-site contains the sequence 5'-AGGTCA-3', although optimal binding of T3 receptors may require additional sequences and the formation of a heterodimeric and not of a homodimeric complex. Furthermore, retinoic acid receptor-binding motifs may play a role in T3-receptor binding (46). Examination of the human collagenase-3 gene revealed four TRE consensus sequences in the region of bp 1,550 to 590 (58). The sequence of the rat collagenase-3 gene is known from bp 456 to +62 and that of the murine gene is not known (51). It is possible that rodent collagenase-3 genes contain TREs in a location similar to that described for the human gene that are responsible for the transcriptional effects observed. However, the exact elements responsible for the T3 effects are not known. T3 receptors may also act by regulating activator protein-1 or -2 sequences, present in the collagenase-3 gene, as it has been reported for the activation and suppression of other genes (20, 49). Examination of the gelatinase B gene promoter reveals the presence of a TRE and activator protein-1 and -2 binding sequences, which might be responsible for the effect of T3 (53). Additional studies will be required to define the exact elements responsible for the induction of collagenase-3 and gelatinase B by T3.
The synthesis of collagenase-1 and -3 by human and rat osteoblasts is regulated by systemic hormones and by cytokines present in the bone microenvironment. Consequently, the apparent constitutive level of collagenase expression by the osteoblast is in fine balance and depends on the exposure of the cell to factors that stimulate and factors that inhibit collagenase synthesis (7, 10, 11, 17, 48, 58, 62, 63). It is possible that T3 interacts with other cytokines, such as interleukin-1 or -6, to enhance collagenase-3 expression as it has been reported for the effects of T3 on bone resorption (59). Because the effect of T3 on MMP expression is slow in onset, we tested whether or not T3 induced the expression of local factors known to stimulate collagenase expression in osteoblasts. Treatment of MC3T3 cells with T3 at 10 nM for 2-72 h did not increase the expression of platelet-derived growth factor BB, fibroblast growth factor 2, IL-1, or IL-6, all known stimulators of collagenase-3 expression (Pereira and Canalis, unpublished observations; Refs. 10, 17, 33, 62, 63). The stimulatory effect of T3 on bone resorption requires the presence of osteoblasts and depends on receptors expressed by osteoblastic cells (6, 65). Consequently, it is not surprising that T3 regulates MMP expression in osteoblasts. T3 was tested at concentrations of 1 nM and higher because previous studies from other investigators revealed that T3 was not effective in cultures of intact calvariae, long bones, and cells of the osteoblastic lineage at lower doses (31, 45, 47, 49). It is possible that under the conditions used in these experiments, concentrations lower than 1 nM could have been effective, although they were not tested. The effects of T3 on MMP expression reported here were observed at 1 nM, and this concentration is ~200-fold higher than the concentrations of free T3 found in the serum of normal humans and 50- to 100-fold higher than the concentrations found in patients with hyperthyroidism (34). The effect of T3 on collagenase and gelatinase expression reported here probably explains the increase in bone collagen degradation that we observed in rat calvariae and may be relevant to its stimulatory actions on bone resorption reported previously (6, 45, 59). It may also explain the increased bone remodeling and increased urinary excretion of collagen cross-links in vivo after thyroid hormone administration and be important in the pathogenesis of the osteopenia after thyroid hormone excess (4, 21, 24). The actions of T3 on MMP expression could have additional effects in bone metabolism because collagenase was recently implied in the fragmentation of IGF-binding protein-5, a binding protein with known stimulatory effects on bone formation (2, 14, 15, 16). Although the activity of these fragments remains to be defined, their appearance in conjunction with an increase in IGF-I might be relevant to selected anabolic actions of T3 in the skeleton (29, 55).
In conclusion, the present studies demonstrate that T3 increases collagenase-3 and gelatinase B expression in osteoblasts by transcriptional mechanisms. It is probable that these effects play a role in the degradation of the collagen matrix and in the osteopenia observed in conditions of thyroid hormone excess.
ACKNOWLEDGEMENTS
We thank Dr. Cheryl Quinn for the rat collagenase cDNA, Dr. Ghislain Opdenakker for the murine gelatinase B cDNA, Dr. Dylan Edwards for the murine TIMP-1, -2 and -3 cDNAs, Dr. Ray Wu for GAPD cDNA, and Dr. John Jeffrey for the rat interstitial collagenase antibody. We also thank Cathy Boucher, Deena Durant, and Sheila Rydziel for technical assistance and Charlene Gobeli and Margaret Nagle for secretarial help.
FOOTNOTES
This work was supported by Grant AR-21707 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. Renata C. Pereira is a recipient of a Fundaç o Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Student Fellowship Grant from the Universidade de Sao Paulo.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. Canalis, Dept. of Research, Saint Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT 06105-1299.
Received 12 February 1999; accepted in final form 29 April 1999.
REFERENCES
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Is FP dying? H-- NO!
IN BEHALF OF FAMILY PRACTICE
We have discussed the fact that experiments have shown that the cytokine cycle and MMPs (matrix metaloproteinases) are involved with a myriad of life process pathology such as cell division to metastatic cancer, normal fast conduction nerve fiber with Nodes of Ranvier to myelin sheath loss and dysfunctional nerve conduction seen in MS, and normal renal function to post impetigo glomerulonephritis with renal dysfunction. The normal blood elements/function to clotting seen in myocardial infarctions (acute cardiac syndrome) and strokes also involve a normal MMP and cytokine cycle seen in the nascent immunologic state to ones that have gone awry . This is seen in strokes and heart attacks and other clotting states. A dysfunctional cytokine cycle seen with the inflammatory cytokines IL1, TNF-alpha an IL6 constitute a 3 man team that if unchecked by itself can lead to clotting disorders.
Yet the normal MMP function (specifically remodeling) could be involved in pathologic processes when the process of remodeling goes unchecked. These processes include MS which is a type of “dysfunctional modeling” involved in autoimmune processes. Today we will explore the question. Can you have a normal cytokine cycle and a purely dysfunctional MMP ? I believe the answer is no. This therefore allows for the possibility of cytokine abnormalities as tumor, MI, stroke and autoimmune disorders’
MARKERS with MMP abnormalities being late markers of disease .
This brings up investment opportunities in cytokine measurement and the possibility that the trained the Family Practitioner will again be the best observer in love for these diseases prior to their needing hospitalization and emergency room visits.
We have discussed the fact that experiments have shown that the cytokine cycle and MMPs (matrix metaloproteinases) are involved with a myriad of life process pathology such as cell division to metastatic cancer, normal fast conduction nerve fiber with Nodes of Ranvier to myelin sheath loss and dysfunctional nerve conduction seen in MS, and normal renal function to post impetigo glomerulonephritis with renal dysfunction. The normal blood elements/function to clotting seen in myocardial infarctions (acute cardiac syndrome) and strokes also involve a normal MMP and cytokine cycle seen in the nascent immunologic state to ones that have gone awry . This is seen in strokes and heart attacks and other clotting states. A dysfunctional cytokine cycle seen with the inflammatory cytokines IL1, TNF-alpha an IL6 constitute a 3 man team that if unchecked by itself can lead to clotting disorders.
Yet the normal MMP function (specifically remodeling) could be involved in pathologic processes when the process of remodeling goes unchecked. These processes include MS which is a type of “dysfunctional modeling” involved in autoimmune processes. Today we will explore the question. Can you have a normal cytokine cycle and a purely dysfunctional MMP ? I believe the answer is no. This therefore allows for the possibility of cytokine abnormalities as tumor, MI, stroke and autoimmune disorders’
MARKERS with MMP abnormalities being late markers of disease .
This brings up investment opportunities in cytokine measurement and the possibility that the trained the Family Practitioner will again be the best observer in love for these diseases prior to their needing hospitalization and emergency room visits.
Tuesday, January 13, 2009
MS and Kidney disease ...... Hope
The following articles/diseases described have something in common...MMP-9
MMP-9 is stabilized by cytokines named IL1 and TNF which are also deemed "inflammatory". They work by promoting a substance called HuR which stabilizes m and t-RNA for MMP.
It appears that IFN has a positive effect in diminishing TNF but the jury is still out on which one (in humans Beta or Gamma).
The hope and take home message is that MMP-9 if diminished will diminish digestion of good kidney mesangial cells and myelin sheaths. Hence we can hope for cures of these dreadful diseases which are more specific.
There also appears to be a tremendous degree of intelligence if you will of the actions of these
actions to hint at their turning on of substances that allow for the proliferation or metastasis
of the original rebellious cell.
Cytokine-mediated modulation of MMPs and TIMPs in multipotential neural precursor cells . Journal of Neuroimmunology , Volume 175 , Issue 1 - 2 , Pages 12 - 18 T . Ben-Hur , Y . Ben-Yosef , R . Mizrachi-Kol , O . Ben-Menachem , A . Miller
Recent studies have implicated the inflammatory process during experimental allergic encephalomyelitis (EAE) in triggering migration and differentiation of transplanted neural precursors cells (NPCs) into the inflamed white matter. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ are key factors in the pathogenesis of brain inflammation in EAE and were shown to enhance NPCs migration in vitro. As cell migration is dependent on extracellular matrix remodeling, involving proteolytic enzyme members of the matrix metalloproteinase (MMPs) family, we characterized the profile of expression of MMPs and their endogenous inhibitors (TIMPs) in rat NPCs, and evaluated the effects of TNF-α, IFN-γ and IFN-β, a clinically proven modulator of brain inflammation, on the expression of these molecules. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+ and NG2(−) cells, which can differentiate into astrocytes, oligodendrocytes and neurons. NPCs' gelatinase activities of MMP-2 and MMP-9, as determined by zymography, were increased by TNF-α, and to a lesser extent by IFN-γ. Semi-quantitative RT-PCR indicated that TNF-α also upregulated MMP-9 mRNA levels. IFN-β suppressed the TNF-α-induced levels of secreted MMP-9 and MMP-2, while enhancing the expression of TIMP-1 and TIMP-2 mRNA. These results suggest that MMPs activity is induced in NPCs by pro-inflammatory cytokines to mobilize them for promoting reparative processes. IFN-β, on the other hand, appears to have an anti-proteolytic influence that may attenuate such NPC-mediated repair processes.
Recent studies have implicated the inflammatory process during experimental allergic encephalomyelitis (EAE) in triggering migration and differentiation of transplanted neural precursors cells (NPCs) into the inflamed white matter. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ are key factors in the pathogenesis of brain inflammation in EAE and were shown to enhance NPCs migration in vitro. As cell migration is dependent on extracellular matrix remodeling, involving proteolytic enzyme members of the matrix metalloproteinase (MMPs) family, we characterized the profile of expression of MMPs and their endogenous inhibitors (TIMPs) in rat NPCs, and evaluated the effects of TNF-α, IFN-γ and IFN-β, a clinically proven modulator of brain inflammation, on the expression of these molecules. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+ and NG2(−) cells, which can differentiate into astrocytes, oligodendrocytes and neurons. NPCs' gelatinase activities of MMP-2 and MMP-9, as determined by zymography, were increased by TNF-α, and to a lesser extent by IFN-γ. Semi-quantitative RT-PCR indicated that TNF-α also upregulated MMP-9 mRNA levels. IFN-β suppressed the TNF-α-induced levels of secreted MMP-9 and MMP-2, while enhancing the expression of TIMP-1 and TIMP-2 mRNA. These results suggest that MMPs activity is induced in NPCs by pro-inflammatory cytokines to mobilize them for promoting reparative processes. IFN-β, on the other hand, appears to have an anti-proteolytic influence that may attenuate such NPC-mediated repair processes.
JBiol Chem. 2003 Dec 19;278(51):51758-69. Epub 2003 Oct
ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR.
Huwiler A, Akool el-S, Aschrafi A, Hamada FM, Pfeilschifter J, Eberhardt W.
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main, Germany.
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR.
Huwiler A, Akool el-S, Aschrafi A, Hamada FM, Pfeilschifter J, Eberhardt W.
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, D-60590 Frankfurt am Main, Germany.
Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
Cytokine-mediated modulation of MMPs and TIMPs in multipotential neural precursor cells . Journal of Neuroimmunology , Volume 175 , Issue 1 - 2 , Pages 12 - 18 T . Ben-Hur , Y . Ben-Yosef , R . Mizrachi-Kol , O . Ben-Menachem , A . Miller
Recent studies have implicated the inflammatory process during experimental allergic encephalomyelitis (EAE) in triggering migration and differentiation of transplanted neural precursors cells (NPCs) into the inflamed white matter. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ are key factors in the pathogenesis of brain inflammation in EAE and were shown to enhance NPCs migration in vitro. As cell migration is dependent on extracellular matrix remodeling, involving proteolytic enzyme members of the matrix metalloproteinase (MMPs) family, we characterized the profile of expression of MMPs and their endogenous inhibitors (TIMPs) in rat NPCs, and evaluated the effects of TNF-α, IFN-γ and IFN-β, a clinically proven modulator of brain inflammation, on the expression of these molecules. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+ and NG2(−) cells, which can differentiate into astrocytes, oligodendrocytes and neurons. NPCs' gelatinase activities of MMP-2 and MMP-9, as determined by zymography, were increased by TNF-α, and to a lesser extent by IFN-γ. Semi-quantitative RT-PCR indicated that TNF-α also upregulated MMP-9 mRNA levels. IFN-β suppressed the TNF-α-induced levels of secreted MMP-9 and MMP-2, while enhancing the expression of TIMP-1 and TIMP-2 mRNA. These results suggest that MMPs activity is induced in NPCs by pro-inflammatory cytokines to mobilize them for promoting reparative processes. IFN-β, on the other hand, appears to have an anti-proteolytic influence that may attenuate such NPC-mediated repair processes.
Recent studies have implicated the inflammatory process during experimental allergic encephalomyelitis (EAE) in triggering migration and differentiation of transplanted neural precursors cells (NPCs) into the inflamed white matter. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ are key factors in the pathogenesis of brain inflammation in EAE and were shown to enhance NPCs migration in vitro. As cell migration is dependent on extracellular matrix remodeling, involving proteolytic enzyme members of the matrix metalloproteinase (MMPs) family, we characterized the profile of expression of MMPs and their endogenous inhibitors (TIMPs) in rat NPCs, and evaluated the effects of TNF-α, IFN-γ and IFN-β, a clinically proven modulator of brain inflammation, on the expression of these molecules. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+ and NG2(−) cells, which can differentiate into astrocytes, oligodendrocytes and neurons. NPCs' gelatinase activities of MMP-2 and MMP-9, as determined by zymography, were increased by TNF-α, and to a lesser extent by IFN-γ. Semi-quantitative RT-PCR indicated that TNF-α also upregulated MMP-9 mRNA levels. IFN-β suppressed the TNF-α-induced levels of secreted MMP-9 and MMP-2, while enhancing the expression of TIMP-1 and TIMP-2 mRNA. These results suggest that MMPs activity is induced in NPCs by pro-inflammatory cytokines to mobilize them for promoting reparative processes. IFN-β, on the other hand, appears to have an anti-proteolytic influence that may attenuate such NPC-mediated repair processes.
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