Comparison of nerve cell and nerve cell plus Schwann cell cultures, with particular emphasis on basal lamina and collagen formation.

The availability of cultures of normal cells (NCs) and Schwann cells (SCs) with and without fibroblasts has allowed us to investigate the sources of endoneurial and perineurial constituents of peripheral nerve. NCs cultured alone, devoid of ensheathment but healthy in appearance, lack basal lamina and extracellular fibrils. In contrast, when SCs accompany NCs, basal lamina and extracellular fibrils are consistently visible around SCs in outgrowth areas formed de novo in culture. These fibrils average 18 nm in diameter, exhibit a repeating banding pattern, and are trypsin-resistant and collagenase-sensitive. Collagen synthesis is also indicated by the incorporation of [14C]proline into peptide-bound hydroxy-proline in NC + SC or SC cultures. That the [14C]hydroxyproline polypeptides formed in NC + SC cultures are collagenous was determined in part by pepsin digestion-ammonium sulfate precipitation-polyacrylamide gel electrophoresis techniques; the 14C-polypeptides migrate to the positions of alpha 1 (I), alpha 2, alpha 1 (III), and alpha B chains of type I, type III, and A-B collagens. Also formed are thin, ruthenium red-preserved strands interconnecting basal laminae. SC ensheathment of axons is similar to that found in the animal; one SC is related to a number of unmyelinated axons or a single myelinated axon. This proclivity to ensheathe and myelinate axons indicates that SC function is not lost during the preparative procedures or after lengthy isolation in culture and provides the most reliable means for SC identification. Perineurial ensheathment and macrophages are lacking in NC + SC culture preparations divested of fibroblasts. We conclude that SCs do not form perineurium or the larger diameter collagen fibrils typical of endoneurium but that in combination with neurons they generate biochemically detectable collagens and morphologically visible basal lamina and thin collagenous fibrils.

Isolated osteoclasts resorb the organic and inorganic components of bone.

Osteoclasts are the principal resorptive cells of bone, yet their capacity to degrade collagen, the major organic component of bone matrix, remains unexplored. Accordingly, we have studied the bone resorptive activity of highly enriched populations of isolated chicken osteoclasts, using as substrate devitalized rat bone which had been labeled in vivo with L-[5-3H]proline or 45Ca, and bone-like matrix produced and mineralized in vitro by osteoblast-like rat osteosarcoma cells. When co-cultured with a radiolabeled substrate, osteoclast-mediated mineral mobilization reached a maximal rate within 2 h, whereas organic matrix degradation appeared more slowly, reaching maximal rate by 12-24 h. Thereafter, the rates of organic and inorganic matrix resorption were essentially linear and parallel for at least 6 d when excess substrate was available. Osteoclast-mediated degradation of bone collagen was confirmed by amino acid analysis. 39% of the solubilized tritium was recovered as trans-4-hydroxyproline, 47% as proline. 10,000 osteoclasts solubilized 70% of the total radioactivity and 65% of the [3H]-trans-4-hydroxyproline from 100 micrograms of 25-50 micron bone fragments within 5 d. Virtually all released tritium-labeled protein was of low molecular weight, 99% with Mr less than or equal to 10,000, and 65% with Mr less than or equal to 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr greater than or equal to 2,000 were examined for the presence of TCA and TCB, the characteristic initial products of mammalian collagenase activity, none was detected by SDS PAGE. In addition, osteoclast-conditioned medium had no collagenolytic activity, and exogenous TCA and TCB fragments were not degraded by osteoclasts. On the other hand, osteoclast lysates have collagenolytic enzyme activity in acidic but not in neutral buffer, with maximum activity at pH 4.0. These data indicate that osteoclasts have the capacity to resorb the organic phase of bone by a process localized to the osteoclast and its attachment site. This process appears to be independent of secretion of neutral collagenase and probably reflects acid protease activity.

Human collagenase: identification and characterization of an enzyme from rheumatoid synovium in culture.

Synovial tissue from patients with rheumatoid arthritis produces lysis of gels of reconstituted collagen fibrils in culture and releases soluble collagenase when cultured in collagen-free medium. Collagen molecules in solution at neutral pH at 20 degrees and 27 degrees C are cleaved by the synovial enzyme into (3/4) and (1/4) length fragments. In this respect the action of synovial enzyme is similar to that of amphibian collagenase and distinct from that of bacterial collagenase. At 37 degrees C reconstituted collagen fibrils and native fibers are attacked by the enzyme and further degraded to polypeptides of low molecular weight. These polypeptides are produced only after denaturation of the larger fragments, which occurs at temperatures near 37 degrees C.

Studies on collagenase from rheumatoid synovium in tissue culture.

Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20 degrees and 27 degrees C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30 degrees C the fragments begin to denature and denaturation is complete at 37 degrees C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37 degrees C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20 degrees -27 degrees C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis.

Studies on purified rheumatoid synovial collagenase in vitro and in vivo.

Rheumatoid synovial collagenase obtained from culture medium can be separated by Sephadex gel filtration into two peaks of enzyme activity. These have been designated as fast-moving and slow-moving rheumatoid synovial collagenases on the basis of their electrophoretic mobility on polyacrylamide gels. The slow-moving rheumatoid synovial collagenase has been highly purified by affinity chromatography on collagen conjugated to Sepharose and used to prepare a monospecific anti-synovial collagenase antiserum. The antiserum against rheumatoid synovial collagenase has permitted the demonstration of immunoreactive collagenase in extracts of rheumatoid synovial tissue that have no detectable enzymatic activity. Collagenase has also been detected immunologically in enzymatically inactive culture medium from the first 24 hr of culture. Recovery of collagenase activity appears to be related to the chromatographic separation of the enzyme from serum antiproteases. The demonstration of collagenase in vivo in rheumatoid synovium adds further support for the concept that the enzyme is present in tissue at levels that are of significance in the pathogenesis of rheumatoid arthritis. In addition, rheumatoid synovial collagenase and human skin collagenase show complete immunologic identity when reacted with monospecific antiserum prepared against either of these purified enzymes, indicating that organ specificity between these two human collagenases is unlikely.

Interstitial collagenase (matrix metalloproteinase-1) expresses serpinase activity.

Human endothelial cells treated with either interleukin-1 beta, tumor necrosis factor-alpha, or phorbol myristate acetate secreted a metalloproteinase that hydrolyzed and inactivated the two major serine proteinase inhibitors (Serpins) found in plasma, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin. Surprisingly, the responsible metalloproteinase was identified as human interstitial collagenase (matrix metalloproteinase-1), an enzyme whose only known physiologic substrate has heretofore been believed to be the extracellular matrix molecule, collagen. The metalloproteinase inactivated the Serpins by cleaving peptide bonds at sites unrelated to those hydrolyzed in collagenous macromolecules. NH2-terminal sequence analysis localized the cleavage sites in the Serpins to regions near their respective reactive site centers at three distinct peptide bonds on the amino-terminal side of bulky, hydrophobic residues. Together, these data indicate that matrix metalloproteinase-1 displays an expanded substrate repertoire that supports the existence of a new interface between connective tissue turnover and Serpin function.

Bone matrix constituents stimulate interleukin-1 release from human blood mononuclear cells.

To test the hypothesis that mononuclear cells are stimulated to release interleukin 1 (IL-1) by bone fragments released in the bone microenvironment during the remodeling cycle, we have investigated the effects of bone matrix and some of its constituents on IL-1 secretin from peripheral blood mononuclear cells (PBMC). Increases in IL-1 activity were observed when either PBMC or adherent monocytes, but not lymphocytes depleted of monocytes, were co-cultured with either human or rat bone particles but not with latex particles of similar size. Co-culture of PBMC with bone particles in a transwell system where the cells were physically separated from the bone particles, or with osteoblast- or osteoclast-covered bone particles, did not stimulate IL-1 release, indicating that a physical contact between PBMC and the bone surface is required for eliciting IL-1 release. This was confirmed by the finding of a lower stimulatory effect of bone particles pretreated with etidronate, a bisphosphonate which decreases the bone binding capacity of PBMC. Constituents of bone matrix, such as collagen fragments, hydroxyproline, and, to a lesser extent, transforming growth factor-beta, but not osteocalcin, alpha 2HS glycoprotein, fragments of either bone sialoprotein or osteopontin, and fibronectin, stimulated PBMC IL-1 release in a dose-dependent fashion. Collagen-stimulated IL-1 release was partially and specifically inhibited by a monoclonal antibody directed against the alpha 2 beta 1-integrin cell surface collagen receptor. These data demonstrate that products of bone resorption, known to be chemotactic for mononuclear cells, stimulate PBMC IL-1 activity. These findings may help explain previous documentation of increased IL-1 secretion by circulating monocytes obtained from patients with high turnover osteoporosis.

A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor.

Fetal membranes usually rupture during the process of labor. Premature fetal membrane rupture occurs not infrequently and is associated with significant fetal and maternal morbidity. The mechanisms of normal and pathologic fetal membrane rupture are not well understood. We have examined structural and biochemical changes in the rat amnion as labor approaches in order to characterize this process in normal pregnancy. Here we report that before the onset of active labor the amnion epithelial cells undergo apoptotic cell death which encompasses degradation of 28S ribosomal subunit RNA and associated P proteins and fragmentation of nuclear DNA. Concurrent with these cellular changes, the amnion type I collagen matrix is degraded with the accumulation of three-quarter length type I collagen fragments in extraembryonic fluid, characteristic of the cleavage of fibrillar collagen by interstitial collagenase. Western blot and immunohistochemical analyses confirmed that interstitial collagenase protein appears in association with the loss of amnion type I collagen. We conclude that amnion epithelial cells undergo a process of programmed cell death associated with orchestrated extracellular matrix degradation which begins before the onset of active labor. Thus, fetal membrane rupture is likely to be the result of biochemical changes as well as physical forces.

The acute phase reactant serum amyloid A (SAA3) is a novel substrate for degradation by the metalloproteinases collagenase and stromelysin.

We found that the matrix metalloproteinases collagenase (MMP-1) and stromelysin (MMP-3) each has the ability to degrade a novel substrate, serum amyloid A (SAA3). SAA3 is a product of rabbit synovial fibroblasts stimulated with phorbol esters or interleukin-1, and it acts in an autocrine or paracrine manner to induce collagenase in both rabbit and human fibroblasts. Recombinant rabbit fibroblast procollagenase and human fibroblast prostromelysin were produced by baby hamster kidney (BHK) cells stably transfected with these genes, and latent enzyme was activated with aminophenylmercuric acetate (APMA). The Km for both enzymes was approximately 10 microM, and the Vmax for collagenase was approximately 6 pmol/minute/100 ng enzyme, while that for stromelysin was about 3-fold faster. Treatment of SAA3 with either enzyme generated a fragment of approx. 6 kDa that has the same amino terminus as the parent molecule, but this fragment was rapidly degraded. We have been unable to isolate C-terminal fragments, suggesting that the mature protein is cleaved at multiple sites and/or that the initial cleavage fragment is readily digested. The amino acid composition of the 6 kDa fragment suggests that the 14 kDa protein is cleaved at residues 50-57, a hydrophobic region that is conserved between rabbit SAA3 and human SAA1. We conclude that the ability of collagenase and stromelysin to degrade SAA3 broadens the repertoire of substrates for these matrix degrading enzymes, and we speculate that the presence of a feedback mechanism that can subvert the autocrine/paracrine stimulation of matrix-degrading enzymes may play a role in limiting matrix degradation during inflammatory conditions.

Hormonal interactions in mammalian collagenase regulation. Comparative studies in human skin and rat uterus.

The production of collagenase by human skin explants in culture is prevented by 10(-8) M dexamethasone, 5 . 10(-4) M dibutyryl cyclic AMP, or 2.5 . 10(-3) M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur. Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10(-7) M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.

Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.

Glucocorticoid modulation of collagenase expression in human skin fibroblast cultures. Evidence for pre-translational inhibition.

Glucocorticoids inhibit collagenase accumulation in the medium of human skin explant cultures. To examine the mechanism for this process, skin fibroblasts were placed in serum-free medium containing various steroids. Dexamethasone produced a dose-dependent inhibition of trypsin-activatable collagenase in the culture medium with maximal inhibition of approx. 85% at 10(-6) M. Dexamethasone failed to inhibit collagenase activity directly. The decrease in activity in the medium was paralleled by a decrease in immunoreactive protein, suggesting inhibition of enzyme synthesis. The specificity of the effect was shown in two ways. At 10(-6) M steroid, only dexamethasone and hydrocortisone were inhibitory; estradiol, progesterone and testosterone produced less than 10% inhibition. In biosynthetic studies, exposure to 10(-7) M dexamethasone for 24 h produced approx. 50% inhibition of collagenase synthesis but caused no greater than 10% inhibition of total protein synthesis. The T1/2 for achieving the effect was approx. 16 h after initial exposure to dexamethasone. These kinetics were parallel to the inhibition caused by actinomycin D and cordycepin, two inhibitors of transcription, but were longer than that caused by cycloheximide (T 1/2 less than 3 h). To examine this process, cells were cultured in the presence or absence of 10(-6) M dexamethasone prior to harvesting mRNA for cell-free translation. In each case the inhibition or enzyme activity in the intact cells was paralleled by a reduction in translatable collagenase mRNA from the same cells. At the same time, there was no significant inhibition of total protein translation by the steroid. These data suggest that glucocorticoids regulate collagenase synthesis at a pre-translational level, possibly through inhibition of transcription.

PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors.

Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway have a key role in the pathogenesis of numerous cancers by altering cell growth, metabolism, proliferation and apoptosis. Interest in targeting the PI3K signaling cascade continues, as new agents are being clinically evaluated. PIK3CA mutations result in a constitutively active PI3K and are present in a subset of pancreatic cancers. Here we examine mutant PIK3CA-mediated pancreatic tumorigenesis and the response of PIK3CA mutant pancreatic cancers to dual PI3K/mammalian target of rapamycin (mTOR) inhibition. Two murine models were generated expressing a constitutively active PI3K within the pancreas. An increase in acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasms (PanINs) was identified. In one model these lesions were detected as early as 10 days of age. Invasive pancreatic ductal adenocarcinoma developed in these mice as early as 20 days of age. These cancers were highly sensitive to treatment with dual PI3K/mTOR inhibition. In the second model, PanINs and invasive cancer develop with a greater latency owing to a lesser degree of PI3K pathway activation in this murine model. In addition to PI3K pathway activation, increased ERK1/2 signaling is common in human pancreatic cancers. Phosphorylation of ERK1/2 was also investigated in these models. Phosphorylation of ERK1/2 is demonstrated in the pre-neoplastic lesions and invasive cancers. This activation of ERK1/2 is diminished with dual PI3K/mTOR inhibition. In summary, PIK3CA mutations can initiate pancreatic tumorigenesis and these cancers are particularly sensitive to dual PI3K/mTOR inhibition. Future studies of PI3K pathway inhibitors for patients with PIK3CA mutant pancreatic cancers are warranted.

Bone-resorbing agents promote and interferon-gamma inhibits bone cell collagenase production.

Parathyroid hormone, prostaglandin E2, 1 alpha,25-dihydroxyvitamin D3, interleukin-1, tumor necrosis factor alpha, and epidermal growth factor, all known stimulators of bone resorption, markedly enhanced collagenase secretion by rat fetus osteoblastlike cells in primary culture as judged by enzyme-linked immunosorbent assay. Untreated cells contained no immunostainable or extractable collagenase. Collagenase was detected in the treated cells and media only after 1-3 h of treatment, and there was no increment in collagenase activity when cells were treated in the presence of actinomycin D or cycloheximide. Cells secreted collagenase in a latent form and also elaborated collagenase inhibitor; chromatographic separation of collagenase from collagenase inhibitor and subsequent activation of the collagenase with trypsin yielded the active species in stimulated but not in unstimulated cells. The ability of individual prostanoids, among seven tested, to promote collagenase production correlated positively with their reported capacity to promote bone resorption. Interferon-gamma (IFN-gamma), a known resorption inhibitor, blocked the increment in collagenase production caused by all agents tested. These results indicate a close linkage between stimulation of bone resorption and collagenase production by osteoblastlike cells. Various resorption stimulators, including some not previously tested for effects on collagenase, augment the de novo synthesis and secretion of collagenase and act by an IFN-gamma-inhibitable mechanism.

Stimulation of collagenase production by rat osteosarcoma cells can occur in a subpopulation of cells.

Recent studies have indicated that neutral collagenase can be produced in bones of rats. In addition, it has been demonstrated by in vitro studies that the enzyme is likely secreted by osteoblasts. Cells of the osteoblastic tumor cell line UMR-106 can be stimulated to produce not only collagenase, but also collagenase inhibitor and plasminogen activator. However, it is conceivable that not all osteoblasts produce all of these proteins. In this study, in which UMR cells were maximally stimulated with PTH, only a subpopulation of cells was observed to produce enhanced levels of collagenase but all cells had the ability to synthesize plasminogen activator. Cells of the rat osteosarcoma line UMR-106-01 were stained for the presence of collagenase and tissue plasminogen activator using an immunohistochemical procedure. In many cases, the cells were exposed to monensin for the final 3 h of incubation as well as to the inducing agent PTH. Monensin prevented export of the enzymes, enabling them to be visualized within their cell or origin. Maximal stimulation of collagenase was demonstrated to occur 8 h after exposure to 10(-8) -10(-7) M PTH. Under these conditions, 14-17% of the cells appeared to synthesize elevated amounts of collagenase (as determined by intense staining). Without PTH stimulation, there was a low level of collagenase in all cells, but less than 1% of the cells stained heavily for the enzyme. In contrast, strong staining for plasminogen activator was observed in all cells with or without PTH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor stimulates expression of interstitial collagenase and inhibitors of metalloproteinases in rat bone cells.

Basic fibroblast growth factor (bFGF) is a bone cell mitogen that affects osteoblastic function by suppressing type I collagen synthesis. The investigators in this study examined whether bFGF also regulates interstitial collagenase and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from 22-day fetal rat calvariae. After exposure to 600 pM bFGF, interstitial collagenase messenger RNA (mRNA) levels, as determined by Northern hybridization analysis, increased after 2 h and were maximally stimulated to approximately 13-fold at 6 h. Exposure of osteoblast-enriched cells to 0.06-6 nM bFGF increased collagenase mRNA in a dose-dependent manner, and bFGF also increased immunoreactive collagenase measured in the culture medium by Western blot analysis. The protein synthesis inhibitor cycloheximide, as well as two inhibitors of protein kinase C, staurosporine and sangivamycin, prevented the bFGF induction of collagenase transcripts, whereas indomethacin, an inhibitor of prostaglandin synthesis, decreased the effect of bFGF on collagenase mRNA levels by about 50%. After exposure to 600 pM bFGF, levels of TIMP 1 and TIMP 3 mRNAs were also maximally stimulated to approximately 6-fold at 16 h and 4-fold at 6 h. bFGF did not modify TIMP 2 expression. In conclusion, bFGF may modulate degradation of collagenous bone matrix by inhibiting collagen as well as stimulating collagenase and TIMPs by osteoblasts.

Regulation of interstitial collagenase expression and collagen degradation by retinoic acid in bone cells.

In osteoblasts, retinoic acid (RA) modulates the synthesis of various proteins, including collagen. However, little is known about the effects of RA on the regulation of interstitial collagenase synthesis and collagen degradation. After treatment of primary osteoblast-enriched (Ob) cells from fetal rat calvariae with 100 nM all-trans-RA (tRA), collagenase mRNA levels, as determined by Northern blotting, did not change after 2 h, increased by 13- to 18-fold after 6 h, and remained elevated until 48 h. Exposure of Ob cells to 10 nM to 1 microM tRA, 13-cis-RA, and 9-cis-RA induced collagenase mRNA in a dose-dependent manner. Collagenase mRNA induction by RA was blocked by cycloheximide. RA increased the stability of collagenase mRNA in Ob cells, suggesting posttranscriptional regulation. Exposure of Ob cells to RA induced immunoreactive procollagenase in medium, as determined by enzyme-linked immunosorbent assay and Western blotting. RA action on collagen degradation was examined in [3H]proline-pulsed intact calvariae chased with and without tRA for 72 h. The release of [3H]hydroxyproline into culture medium was increased by 64% in the presence of 10 nM to 1 microM tRA. In conclusion, RA increases collagenase synthesis and collagen degradation in bone and is likely to play an important role in bone remodeling.

Insulin-like growth factors inhibit interstitial collagenase synthesis in bone cell cultures.

Insulin-like growth factor-I (IGF-I) and IGF-II are among the most prevalent growth factors secreted by bone cells and are presumed to act as autocrine regulators of bone formation. We recently demonstrated that IGFs inhibit bone collagen degradation, and we postulated that they may either inhibit the expression of interstitial collagenase or stimulate the synthesis of tissue inhibitors of metalloproteinase-1 (TIMP-1), -2, or -3. We tested the effects of IGF-I and -II on collagenase and TIMP-1, -2, and -3 expression in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells). Steady state messenger RNA (mRNA) levels were determined by Northern blot analysis, and collagenase concentrations were determined in the culture medium by a specific immunoassay. After 2-6 h of treatment, IGF-I and -II decreased collagenase transcripts by up to 80%. IGF-I was a more potent inhibitor than IGF-II, because it was active at doses as low as 10 nM, whereas a dose of 100 nM was required to observe the IGF-II effect. In addition, IGF-I and -II opposed the stimulatory effect of retinoic acid on collagenase transcripts. Immunoreactive collagenase levels were not detectable in control or IGF-treated cultures, but IGF-I and -II decreased the levels induced by retinoic acid by 70-90%. The protein synthesis inhibitor cycloheximide superinduced collagenase transcripts, and IGF-I or -II decreased this mRNA induction to levels similar to, but not lower than, those observed in control cultures. The effects of IGF-I and -II on collagenase transcripts were not modified by the DNA synthesis inhibitor hydroxyurea at 1 mM. Neither IGF-I nor IGF-II modified the expression of TIMP-1, -2, or -3 mRNA in Ob cells. TIMP protein levels were not determined, and our study does not exclude a translational or posttranslational effect of IGF. In conclusion, IGF-I and -II decrease interstitial collagenase transcripts as well as induced protease levels in Ob cells, and this effect may contribute to their inhibitory actions on bone collagen degradation.

Transcriptional and posttranscriptional regulation of interstitial collagenase by platelet-derived growth factor BB in bone cell cultures.

Platelet-derived growth factor (PDGF), a bone cell mitogen, stimulates bone collagen degradation and does not enhance bone matrix apposition rates. The mechanism of the effect on collagen degradation is unknown, and it could involve changes in interstitial collagenase synthesis. We tested the effects of PDGF on interstitial collagenase expression in cultures of osteoblast-enriched cells from fetal rat calvariae (Ob cells). After 4-8 h of treatment, PDGF BB at 0.3 nM increased steady state collagenase messenger RNA (mRNA), whereas PDGF AA had no effect. The effect of PDGF BB on collagenase transcripts was dose dependent. PDGF BB increased the levels of immunoreactive collagenase after 6 h, whereas the levels were decreased after 16 h. Stimulation of collagenase mRNA by PDGF BB was dependent on de novo protein synthesis and activation of protein kinase C. PDGF BB prolonged the half-life of collagenase mRNA in transcriptionally arrested cells. PDGF BB initially increased and subsequently decreased the rate of collagenase gene transcription and the levels of collagenase heterogeneous nuclear RNA. In conclusion, PDGF BB regulates interstitial collagenase in Ob cells by transcriptional and posttranscriptional mechanisms, and this effect may contribute to its stimulatory actions on bone collagen degradation.