Induction Of Collagenase Research Articles

Serotonin-mediated Production of Interstitial Collagenase by Uterine Smooth Muscle Cells Requires Interleukin-1α, but not Interleukin-1β

The activation of the gene for interstitial collagenase in myometrial smooth muscle cells is absolutely dependent upon the presence of serotonin. Our previous studies investigating the mechanisms of this induction demonstrated that the mRNAs of both interleukin-1 (IL-1) isoforms, IL-1alpha and IL-1beta, are induced by serotonin and that the induction of IL-1 is required for the subsequent induction of collagenase. These data provided compelling evidence that serotonin-induced IL-1 acts via an autocrine loop in activating the collagenase gene. The experiments described here were designed to examine the potential role of each IL-1 isoform in collagenase production by using neutralizing antisera specific to each isoform of the cytokine. The antisera were examined for their ability to inhibit the serotonin-dependent production of the mRNA for collagenase and of the cytokines themselves. Neutralizing antiserum against IL-1alpha, but not against IL-1beta, inhibited the induction of the mRNA for collagenase and of the mRNAs for both IL-1alpha and IL-1beta. Western analysis indicated that detectable levels of IL-1alpha protein, but not that of IL-1beta, are produced at the time of serotonin-dependent collagenase induction. In contrast, significant levels of IL-1beta protein are detected only when bacterial lipopolysaccharide is added to the cells. Taken together, the results of our study indicate that IL-1alpha, but not IL-1beta, plays an obligatory role in multiple serotonin-mediated gene regulations in the myometrial smooth muscle cell. In addition, the data suggest that IL-1beta production has the potential for modifying myometrial function in pathological settings, particularly that of uterine infection.

Interleukin-4 suppression of interleukin-1-induced transcription of collagenase (MMP-1) and stromelysin 1 (MMP-3) in human synovial fibroblasts.

To determine the effects of interleukin-4 (IL-4) on IL-1 induction of collagenase (matrix metalloproteinase 1 [MMP-1]) and stromelysin-1 (MMP-3) in human synovial fibroblasts. Northern blot analysis was performed to determine the effects of IL-4 on IL-1 induction of MMP messenger RNA (mRNA). MMP protein levels were determined by enzyme-linked immunosorbent assay, and prostaglandin E2 (PGE2) levels were measured by enzyme immunoassay. Run-on transcription assays and transient transfection experiments were performed to determine whether the effects of IL-4 occur at the level of transcription. Activator protein 1 (AP-1) binding was assessed by electrophoretic mobility shift assay. Northern blot analysis revealed that coincubation of synovial fibroblasts with IL-1 and IL-4 resulted in a significant decrease in both collagenase and stromelysin mRNA levels compared with IL-1 alone, with a concomitant decrease in MMP protein levels. This inhibition is dose dependent, with an IC50 (50% inhibition concentration) for both MMPs of approximately 0.3 ng of IL-4 per ml, and is at least somewhat selective, since IL-1 induction of c-fos mRNA is not affected. Nuclear run-on experiments and transient transfection studies demonstrated that the suppression of IL-1-induced collagenase and stromelysin expression by IL-4 occurs at least in part at the transcriptional level, and that binding of transcription factor AP-1 is not affected. Although IL-1-induced levels of PGE2 are reduced by IL-4, exogenous addition of PGE2 does not abrogate the inhibitory effects of IL-4 on MMP expression. IL-4 inhibits IL-1 induction of both collagenase and stromelysin, as well as PGE2 production, in human synovial fibroblasts. The inhibition occurs at least in part at the level of transcription, does not affect binding of transcription factor AP-1, and appears to involve a mechanism that is independent of the ability of IL-4 to inhibit production of PGE2.

Serotonin regulates the expression of the gene for α2-macroglobulin in myometrial smooth muscle cells

Serotonin (5-hydroxytryptamine (5-MT)) has been shown to be a regulator of gene expression in rat myometrial smooth muscle cells (SMC). Serotonin activates the genes for interstitial collagenase, interleukin-1 α, -1 β and interleukin-6, among others. On the other hand, serotonin down-regulates the genes for types I and III collagen and fibronectin. Here we show that serotonin is also a negative regulator of the expression of anti-protease α 2-macroglobulin ( α 2M) in SMC. The serotonin-dependent repression occurs at both the mRNA and protein levels, and is mediated by the 5-HT 2A receptor subtype. The inhibitory effect is prevented by cycloheximide, indicating the requirement for the synthesis of one or more proteins. Interleukin-1 (IL-1), which is induced by serotonin in SMC and is required for subsequent interstitial collagenase induction, appears not to be one of these intermediates. On the other hand, progesterone, the major steroid hormone of pregnancy, is capable of reversing the serotonin-mediated inhibition of α 2M. The phorbol myristate acetate (PMA), which mimics the induction of interstitial collagenase by serotonin, fails to affect the inhibition of α 2M production. The cell-permeable cyclic AMP analogue 8-bromoadenosine 3′:5′-cyclic monophosphate sodium salt (8-bromo-cAMP), is, however, capable of fully reproducing the action of serotonin on α 2M. These results further speak to the ability of serotonin to regulate gene expression in the myometrial SMC, both positively and negatively. In addition, although all the effects of serotonin so far identified are mediated by the 5-HT 2A receptor, different post-receptor pathways appear to mediate the positively and negatively regulated genes.

UVA-1 phototherapy improves morphea (localised scleroderma) by induction of interstitial collagenase (MMP-1)

UVA-1 phototherapy improves morphea (localised scleroderma) by induction of interstitial collagenase (MMP-1)

Ets-1 is an early response gene activated by ET-1 and PDGF-BB in vascular smooth muscle cells.

Ets-1 is a transcription factor that activates expression of matrix-degrading proteinases such as collagenase and stromelysin. To study the control of ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to regulate VSMC migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of ets-1 mRNA. These effects were abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic PMA treatment. Ets-1 mRNA was superinduced by PDGF-BB and ET-1 in the presence of cycloheximide. The chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the depletion of endoplasmic reticulum intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin inhibited PDGF-BB- and ET-1-induced ets-1 mRNA, whereas ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid had no effect. However, [Ca2+]i release alone was not sufficient to increase ets-1 mRNA. Forskolin blocked ET-1-, PDGF-BB-, and PMA-induced ets-1 mRNA, as well as inositol phosphate formation, consistent with an effect through impairment of PKC activation. Inhibitors of ets-1 gene expression, such as H-7 and herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of vascular remodeling after arterial injury.

Induction of interstitial collagenase (MMP-1) by UVA-1 phototherapy in morphea fibroblasts

Induction of interstitial collagenase (MMP-1) by UVA-1 phototherapy in morphea fibroblasts

Parathyroid allotransplantation without immunosuppression

The hypocalcaemia of permanent hypoparathyroidism is a severe and sometimes fatal complication after thyroid and parathyroid surgery. Due to the multiple metabolic functions of parathyroid hormone, the symptoms and associated diseases of hypoparathyroidism cannot always be controlled sufficiently with oral calcium and vitamin D supplements. We report the successful treatment of two patients with postoperative hypoparathyroidism by allotransplantation of cultivated parathyroid tissue particles that were encapsulated with a semipermeable membrane (microencapsulation). A 52-year-old woman and a 55-year-old woman had postoperative hypoparathyroidism after subtotal bilateral thyroid resection for multinodular goitre. To relieve symptoms the hypocalcaemia was treated with intravenous calcium (8 g and 4 g calcium per day, respectively) and high-dose oral vitamin D. Intact parathyroid hormone was not detectable in the serum of the two patients. Parathyroid allotransplantation was considered because both patients suffered from tetany, bone pain, depression, and impaired vision. Previous studies showed that MHC class I and II antigens are expressed on parathyroid tissue and their concentrations might determine graft outcome. Reduction of MHC expression can be achieved by depletion of immunogenic MHC-bearing cells with magnetic microspheres or tissueculture passages. The combination of microencapsulation and tissue-culture passage provides the key for successful isotransplantation, allotransplantation, and xenotransplantation of parathyroids as shown in long-term animal studies. The donor for both patients was an ABO compatible, HLA-mismatched patient with parathyroid hyperplasia due to secondary hyperparathyroidism. After parathyroidectomy, the tissue was cut into pieces of 1 mm. These particles were immersed in amitogenic degassed 2% sodium-alginate. The alginate was configured to allow nutritive substances as well as parathyroid hormone to pass through the membrane, while excluding immunogens and mediators of the cellular immune response. The suspension was passed through a spray nozzle (inner diameter 2 mm) for encapsulation with a constant flow of 6·5 L/min. The resulting spheres (microcapsules) were hardened by a 7-min incubation in 10 mmol/L barium chloride. Following multiple washes, microcapsules were cultured (RPMI 1640 with glutamine, 10 000 μg/mL streptomycin and penicillin, 10% fetal calf serum, and 2·4 mmol calcium chloride) for 2 days. 20 microcapsules were then transplanted into the brachioradial muscle of the non-dominant forearm. The study protocol was approved by the ethics committee of the Philipps-University of Marburg and written informed consent was given by both patients. The serum concentrations of total calcium and intact parathyroid hormone (iPTH) in one of the patients before and after allotransplantation are presented in the figure. The serum values of calcium and iPTH of the other case showed similar increases. For both patients calcium and vitamin D replacement therapy was reduced by half daily, beginning the first day after allotransplantation. They were discharged from the hospital without any hypocalcaemic symptoms and with no immunosuppressive therapy. The oral calcium and vitamin D replacement therapy was 0·6 g and 0·25 μg daily, in both patients, respectively. The combination of tissue-culture passage and microencapsulation could make parathyroid allotransplantation a practical option for successful treatment completely (table). No side effects were observed. Palpation showed softening of sclerotic lesions. Histopathological analysis of skin biopsy specimens taken 18 h after the last UVA-1 irradiation of this treatment course revealed dermal collagen with a thickness close to that of normal skin. ISH was done on formalin-fixed and paraffin-embedded tissue specimens with human interstitial collagenase-specific mRNA transcript. The probe sequence (5'dGGA AGC CAA AGG AGC TGT AGA TGT CCT-3') was unique when compared with all primate nucleic-acid sequences registered in the Entrez database. For IHC a monoclonal mouse serum (antibody 41-IE5, Oncogene Research Products, Cambridge, MA, USA) specific for human MMP-1 protein was used and immunohistochemically labelled. Both assays produced colorimetric reactions of variable intensity (table). In pretreatment specimens, a specific colorimetric signal was either absent (–) or weak (+) in fibroblastic cells. After UVA-1 treatment, an increase of interstitial collagenase m-RNA and protein expression was detected separately in most dermal fibroblastic cells. Here, a moderate (++) to strong (+++) signal was identified either by ISH, IHC, or both in all tissue specimens. In two of the lesions, signal intensity was greatest in fibroblasts of the upper papillary dermis. In both assays, UVA-1 phototherapy induced an increased signal for interstitial collagenase, weaker than that seen in fibroblasts, mainly in the upper layer of keratinocytes, melanocytes, and endothelial cells. Our results show that UVA-1 irradiation indeed induces expression of interstitial collagenase in fibroblasts of sclerotic morphea plaques. Together with the clinical and histological improvement of skin lesions, this is evidence that UVA-1 phototherapy acts by induction of interstitial collagenase in the clearance of morphea. In other studies, UVA-1 phototherapy has been shown to induce a variety of cytokines and soluble factors thereby causing profound immunomodulation. Our results also suggest that UVA-1 irradiation may also be of therapeutic benefit in other conditions characterised by excessive collagen production including keloid scars and a variety of rheumatological diseases.

Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1β (IL-1β) in human fibroblasts and BC-8701 breast cancer cells

Interleukin-1 beta (IL-1 beta) is a potent cytokine that stimulates interstitial collagenase-1 (matrix metalloproteinase-1; MMP-1). In this study, we compared the mechanism(s) by which IL-1 beta induces collagenase gene expression in two very different cells, normal human foreskin fibroblasts (HFFs) and an aggressive breast cancer cell line, BC-8701 cells. Northern analysis showed that the time course of collagenase induction was distinct in the two cells: although both cells expressed low levels of MMP-1 constitutively, addition of IL-1 beta increased MMP-1 mRNA in HFFs by 1 h and levels remained high over a 24-h period. In contrast, MMP-1 levels in IL-1 beta-treated BC-8701 cells did not increase until 4 h, peaked by 12 h and then declined. To analyze the transcriptional response, we cloned and sequenced more than 4,300 bp of the human MMP-1 promoter, and from this promoter clone, we prepared a series of 5'-deletion constructs linked to the luciferase reporter and transiently transfected these constructs into both cell types to measure both basal and IL-1 beta induced transcription. When both cell types were uninduced, promoter fragments containing less than 2,900 bp gave only a minimal transcriptional response, while larger fragments showed increased transcriptional activity. With IL-1 beta treatment, significant responsiveness (P < 0.001) in HFFs was seen only with the larger fragments, while in the BC-8701 cells, all fragments were significantly induced with IL-1 beta. Finally, we found that IL-1 beta stabilized MMP-1 mRNA in normal fibroblasts, but not in BC-8701 breast cancer cells. We conclude that both the transcriptional and post-transcriptional regulation of MMP-1 gene expression by IL-1 beta is controlled by cell-type specific mechanisms, and we suggest that IL-1 induced MMP-1 expression in tumor cells and in neighboring stromal cells may amplify the invasive ability of tumor cells.

Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro

Ultraviolet (UV) A irradiation of human dermal fibroblasts elicits an increase in specific mRNA amounts and bioactivities of the cytokines IL-1α, IL-1β, and IL-6. These effects are enhanced in deuterium oxide-based medium and are diminished in the presence of non-toxic concentrations of sodium azide. Furthermore, generating singlet oxygen outside the cells by irradiation of rose bengal-coated resin particles with visible light ( λ>450 nm) results in the induction of interstitial collagenase, IL-1 and IL-6, similar to the response observed with UVA irradiation. These observations suggest that singlet oxygen is an early intermediate in the signaling pathway of IL-1 and IL-6 mediating UVA induction of interstitial collagenase (E.C. 3.4.24.7). Furthermore, singlet oxygen appears to initiate this complex UV response at the cell membrane.

High-dose UVA 1 radiation therapy for localized scleroderma

Fibrotic skin lesions in patients with localized scleroderma can cause muscle atrophy, disfigurement, and flexion contractures. There is no effective therapy for this disease. Skin fibrosis is thought to be caused by decreased collagenase activity. Collagenase activity can be induced in dermal fibroblasts by UVA1 irradiation. Our purpose was to assess whether UVA1 radiation therapy is effective for patients with localized scleroderma. Patients with localized scleroderma (n = 17) were exposed 30 times to 130 J/cm2 UVA1 (high-dose UVA1 therapy; n = 10) or 20 J/cm2 UVA1 (low-dose UVA1 therapy; n = 7). Therapeutic effectiveness was assessed by evaluation of (1) clinical features, (2) thickness of sclerotic plaques, and (3) cutaneous elastometry. Sequential biopsy specimens from treated lesions were analyzed for collagenase I messenger RNA (mRNA) expression by semiquantitative reverse transcriptase-polymerase chain reaction. In all patients, high-dose UVA1 therapy softened sclerotic plaques, and complete clearance was observed in four of 10 patients. High-dose UVA1 therapy significantly reduced thickness and increased elasticity of plaques. These changes could not be detected in unirradiated control plaques and were still present in 9 of 10 patients 3 months after cessation of therapy. For all factors assessed, high-dose UVA1 was superior to low-dose UVA1 therapy (p = 0.001). High-dose UVA1 therapy increased collagenase I mRNA expression about 20-fold in treated plaques. High-dose UVA1 therapy is effective in the treatment of localized scleroderma. Effectiveness is UVA1 dose dependent and is associated with induction of collagenase I expression.

Interleukin-6 and Its Soluble Receptor Cause a Marked Induction of Collagenase 3 Expression in Rat Osteoblast Cultures

Interleukin-6 (IL-6), a cytokine produced by skeletal cells, increases bone resorption, but its effects on collagenase expression are unknown. We tested the effects of IL-6 and its soluble receptor on collagenase 3 expression in osteoblast-enriched cells from fetal rat calvariae (Ob cells). IL-6 caused a small increase in collagenase mRNA levels, but in the presence of IL-6-soluble receptor (IL-6sR), IL-6 caused a marked increase in collagenase transcripts after 2-24 h. In addition, IL-6sR increased collagenase mRNA when tested alone. IL-6 and IL-6sR increased immunoreactive collagenase levels. Cycloheximide and indomethacin did not prevent the effect of IL-6 and IL-6sR on collagenase mRNA levels. IL-6 and IL-6sR did not alter the decay of collagenase mRNA in transcriptionally arrested Ob cells and increased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. IL-6 and IL-6sR increased collagenase 3 mRNA in MC3T3 cells but only modestly in skin fibroblasts. IL-6 and IL-6sR enhanced the expression of tissue inhibitor of metalloproteinases 1. In conclusion, IL-6, in the presence of IL-6sR, increases collagenase 3 synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the action of IL-6 on bone matrix degradation and bone resorption.

Integrin α2β1-Dependent Contraction of Floating Collagen Gels and Induction of Collagenase Are Inhibited by Tyrosine Kinase Inhibitors

A cell culture inside a three-dimensional gel of fibrillar collagen is an experimental model used to study the response of cells to the extracellular matrix. Many cell types induce the contraction of gel and simultaneously decrease their production of type I collagen, whereas the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is enhanced. We have previously shown that in osteogenic cells the collagen receptor α2β1 integrin is a positive regulator of MMP-1 and that the number of α2β1 integrins on the cell surface also regulates the magnitude of contraction. However, the downregulation of collagen mRNA levels is not initiated by α2β1 integrin. Here, we have studied in human KHOS-240 and MG-63 osteosarcoma cells and in human skin fibroblasts the effects of tyrosine kinase inhibitors on collagen gel contraction and on the regulation of MMP-1 and collagen α1(I) genes by extracellular collagen. The induction of MMP-1 could be inhibited by all tyrosine kinase inhibitors tested with the exception of genistein. None of them could prevent the downregulation of collagen expression. Thus, the collagen-induced alterations in the expression of MMP-1 and collagen α1(I) seem to be dependent on distinct signal transduction pathways. Many of the inhibitors, including genistein, could prevent the contraction of collagen gels. The effect was not related to their ability to inhibit cell growth, because an inhibitor specific for DNA synthesis and cell division did not have the same effect. Thus, we suggest that the process of collagen gel contraction requires protein-tyrosine phosphorylation and that the ability of cells to contract collagen gels is not related to the induction of MMP-1 or to the level of collagen α1(I) expression. Finally, we propose that the tyrosine kinase inhibitors might be considered as candidate molecules in the treatment of pathological scar contraction.

Cytokine Control of Interstitial Collagenase and Collagenase-3 Gene Expression in Human Chondrocytes

Human collagenase-3 expression, previously seen only in a breast tumor tissue, is here shown to be expressed in primary human chondrocytes derived from the joint tissue and in transformed human chondrocytes. Its mRNA is inducible by the inflammatory cytokines interleukin-1beta plus tumor necrosis factor-alpha, but not by phorbol 12-myristate 13-acetate and only slightly by the growth factors platelet-derived growth factor and epidermal growth factor. Human synovial fibroblasts, another prominent cell type in the joint tissue, do not produce collagenase-3 message. Expression of the murine collagenase, which is possibly the counterpart of human collagenase-3, is induced by interleukin-1beta plus tumor necrosis factor-alpha, and its full induction requires the presence of the transcription factor, c-FOS. This family of transcription factors also plays a role in induction of human collagenase-3, since it binds to the AP-1 site of this matrix metalloproteinase.

Parathyroid hormone induction of rat interstitial collagenase mRNA in osteosarcoma cells is mediated through an AP-1-binding site.

Interstitial collagenase is secreted by the osteoblast in response to bone-resorbing agents. Previously, we cloned the rat interstitial collagenase cDNA from UMR 106-01 rat osteoblastic osteosarcoma cells. We demonstrated that induction of collagenase by PTH, a powerful resorbing agent, in UMR 106-01 cells is in part transcriptional. In the present study we isolate and characterize the rat interstitial collagenase gene. The gene consists of 10 exons and spans approximately 12 kbp. The major transcriptional start site, determined by primer extension analysis and confirmed by RNase protection assay, is 25 nucleotides upstream of the translational start site. The previously isolated cDNA was missing the 5'-untranslated sequence in addition to 17 nucleotides of the signal sequence of the preproenzyme; therefore, we also present these data. Chloramphenicol acetyl transferase (CAT) analyses were performed on the 5'-upstream region of the gene. These data indicate that PTH appears to mediate its effect through an AP-1 consensus-binding sequence (-51). Footprint analysis demonstrates protein binding to this site. Site-specific mutagenesis markedly decreased protein binding, which correlated directly with a decrease in CAT activation by PTH. Supershift data indicate that cAMP response element binding protein (CREB) is binding to this AP-1 consensus sequence. In addition we demonstrate that PTH induces phosphorylation of CREB.

The induction of interstitial collagenase is associated with catabolism of type I collagen in the rat amnion at term

The induction of interstitial collagenase is associated with catabolism of type I collagen in the rat amnion at term

H-89 inhibits collagenase induction by phorbol ester through a mechanism that does not involve protein kinase A

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) regulates the activity of growth-factor-induced pathways at the level of cytoplasmic kinases and nuclear transcription factors. We observed that H-89, an inhibitor of PKA, induced mitogen-activated protein (MAP) kinase activity in a 12V- ras-transformed fibroblast cell line. In contrast, H-89 inhibited phorbol-ester-mediated induction of MAP kinase, junB messenger ribonucleic acid (mRNA), and collagenase mRNA in these cells. Phorbol-ester stimulation of a collagenase-promoter reporter construct was also inhibited by H-89. However, stimulation of the collagenase promoter was not inhibited by overexpression of the PKA-inhibitory protein PKI. These data suggest that H-89 inhibits the activity of an enzyme required for phorbol-ester induction of collagenase mRNA, but that this inhibition does not occur at the level of PKA.

Fish oil may impede tumour angiogenesis and invasiveness by down-regulating protein kinase C and modulating eicosanoid production

Inhibition of angiogenesis shows considerable promise as a strategy for treating solid malignancies. Induction of collagenase by protein kinase C plays an important role in the angiogenic process as well as in metastasis. Lipoxygenase products are required for endothelial cell mitosis, and also promote collagenase production. By down-regulating hormonal activation of protein kinase C and modulating eicosanoid metabolism, ingestion of omega-3-rich fish oils may impede angiogenesis and reduce tumor invasiveness -thus rationalizing the growth-retardant and anti-metastasic effects of fish oil feeding almost invariably seen in animal tumour models. Certain other anti-inflammatory agents - including cromolyn (an inhibitor of protein kinase C activation) and gamma-linolenic acid (which indirectly inhibits lipoxygenase) may have analogous tumour-retardant activity. Clinical application of supplemental fish oil in cancer therapy is long overdue.

Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B.

The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting beta-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology, PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.

Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.

Fos is an essential component of the mammalian UV response.

Mouse 3T3 fibroblasts lacking c-fos were employed to demonstrate an essential function of the UV-inducible transcription factor AP-1 (Fos/Jun) in the response to the cytotoxic effects of short-wavelength ultraviolet (UVC) radiation. Clonogenic survival and proliferation of cells lacking c-fos were drastically reduced following UV irradiation. This UV hypersensitivity manifests itself primarily in increased cell death, partly by apoptosis, and prolonged recovery time from UV-induced cell cycle arrest. Co-culture with wild-type cells did not ameliorate the hypersensitivity of mutant cells. Transcriptional induction of the c-Fos target genes collagenase I, stromelysin-1 and stromelysin-2 by UV is almost absent in cells lacking c-fos which correlates with a reduced UV induction of AP-1 DNA-binding and transactivation activity. The repair of UV-induced DNA lesions was not affected, as shown by unscheduled DNA synthesis and host cell reactivation assays. These data demonstrate that c-Fos is involved in a novel protective function other than DNA repair against the harmful consequences of UVC.