Increase In Collagenase Research Articles

Role of Age‐Dependent Changes in MMP Expression in Acute Kidney Injury

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism(s) underlying the increased susceptibility to injury remain undefined. Given the emerging role of MMPs in ischemic renal injury, the renal mRNA expression of MMPs and their endogenous inhibitors, TIMPs (tissue inhibitor of metalloproteinases), were also examined in young, aged‐AL and aged‐CR rats. Interestingly, age‐dependent increases in the expression of MMP‐7, ‐9, ‐13, and ‐14 were seen, and these changes were attenuated by caloric restriction. The expression of TIMP‐1, but not TIMP‐2 or ‐3, was increased by age and attenuated by caloric restriction. Changes in MMP expression gene expression were paralleled by an age‐dependent increase in collagenase, gelatinase, and membrane type‐MMP activity. Specific increases in MMP‐7, ‐9, and ‐13 activity was seen in urine and kidney tissue, respectively. These results suggest an association between the age‐related increase in susceptibility to injury and overexpression of select MMPs (‐7, ‐9, ‐13, and ‐14) in the kidney, implying a therapeutic potential for inhibition of these MMPs in acute kidney injury.

Direct measurement of collagenase in colonic anastomosis

Collagenase has been implicated in colonic anastomotic dehiscence but the enzyme has not previously been specifically measured in colonic healing. A 72 h tissue culture method for colonic tissue and a radiochemical assay for collagenase were adapted to measure the enzyme in healing rabbit colon, with specificity of the assay confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis. Normal and postoperative colon secreted collagenase, predominantly in a latent form, in the first 24 h of culture. Total activity reached a plateau after 48 and 72 h in culture, when 50-70 per cent of the enzyme was in an active form. At these times in culture, activity was significantly higher than after 24 h (P less than 0.001). One day after anastomosis the total amount of collagenase secreted in culture was higher than normal but the increase did not achieve significance. Three days after anastomosis the colon secreted more collagenase than explants from 1 day postoperative tissue (P less than 0.002). The proportion of active enzyme in the first 24 h in culture was also increased. Since active collagenase can be measured in culture medium from both normal and postoperative colon, the tissue may be secreting plasminogen activator which allows plasmin to activate the enzyme. The increase in collagenase after operation coincided with a decrease in collagen concentration in the colon wall, measured by hydroxyproline. This supports previous suggestions that collagenase contributes to anastomotic dehiscence. However, the findings must be interpreted with caution as the variance of the results was shown to be predominantly due to time in culture, suggesting this could be a bigger influence than the operation itself. In addition, our previously reported immunohistochemical study of this system indicated that collagenase only occurred in a localized region, restricted to the everted portion of the anastomosis, with the activity being tightly controlled by its inhibitor, tissue inhibitor of metalloproteinases.

Extracellular 14-3-3σ Protein: A Potential Mediator of Epithelial–Mesenchymal Interactions

Since its identification as a target of the p53 transcription factor, the 14-3-3 gene has attracted considerable attention (Hermeking et al, 1997). As 14-3-3 is epigenetically silenced in many carcinomas, it is regarded as a tumor-suppressive gene (reviewed inHermeking, 2003). Experimental removal of the 14-3-3 gene leads to an inability to maintain a stable G2/M-arrest after DNA-damage and sensitizes cells to DNA-damaging treatments commonly used in cancer therapy (Chan et al, 1999;Lodygin et al, 2004). 14-3-3 is also known as STRATIFIN/SFN, a name related to its high expression in stratifying keratinocytes, where it was identified first (Leffers et al, 1993). The 14-3-3 proteins regulate numerous cellular processes in all multi-cellular species analyzed. In humans 7 isoforms of 14-3-3 proteins have been described, which form dimers that bind to protein ligands following serine/threonine-phosphorylation of two canonical 14-3-3-binding motifs, R(S/X)XpSXP and RXXXpSXP, where pS represents phospho-serine or phospho-threonine and X any amino acid. Association with 14-3-3 proteins regulates the function of ligands by inter- and intra-compartmental sequestration, activation/inactivation of enzymatic activity and promotion/inhibition of protein interactions (reviewed inHermeking, 2003).

Suggested mechanisms of action of UVA phototherapy in morphea: a molecular study.

Ultraviolet A (UVA) phototherapy proved to be an efficient line of treatment of scleroderma. The mechanism through which it acts is still not clear. To detect the mechanism of action of UVA phototherapy in morphea through measuring its effect on the levels of different parameters related to collagen metabolism. Twenty-one cases of morphea were treated with low-dose broad-band UVA for 20 sessions. Twelve cases received 20 J/cm(2)/session with a cumulative dose of 400 J/cm(2) and nine cases received 10 J/cm(2)/session with a cumulative dose of 200 J/cm(2). The response was assessed clinically every week. Two skin biopsies were taken from the lesional skin of each patient before starting and after the end of therapy. Paraffin sections were examined for quantitative polymerase chain reaction measurement of collagen I, collagen III, collagenase, transforming growth factor-beta (TGF-beta) and interferon gamma (IFNgamma). Eighteen patients reported remarkable softening of the skin lesions, with variable degrees ranging from moderate in 57.1% of them good in 19% to very good response in 9.5%. After treatment, all the studied parameters revealed statistically significant changes. There was a significant decrease in collagen I, collagen III and TGF-beta and a significant increase in collagenase (MMP-1) and IFNgamma. The relative change was found to be greatest in collagenase, followed by IFNgamma then TGF-beta and finally collagen I. The changes in collagen I, collagenase, IFNgamma and TGF-beta were found to increase gradually with the degree of clinical response. In all the parameters studied the relative change was significantly higher in cases treated with 20 J/cm(2)/session in contrast to those treated with 10 J/cm(2)/session although no statistically significant difference could be detected in the clinical response to those doses. The efficacy of low-dose UVA phototherapy in the treatment of localized scleroderma is mainly obtained by the increased production of MMP-1 and IFNgamma, and to a lesser extent by decreasing TGF-beta and collagen production. Concerning the use of 10 or 20 J/cm(2)/session those effects are dose dependent, but the clinical response does not significantly differ.

Full Thickness EpiDerm™: A Dermal–Epidermal Skin Model to Study Epithelial–Mesenchymal Interactions

Unlike previous dermal-epidermal models, Full Thickness EpiDerm is cultured in an easily manipulated cell culture insert, and the tissue extends from wall to wall. In terms of ease of use, these characteristics greatly facilitate the testing of potential allergens or irritants in that direct topical application is possible. Topical exposure to the common surfactant, 1% Triton X-100, resulted in MTT tissue viability dose-response curves that fell within the normal range of the keratinocyte-only tissue, EpiDerm. Currently, in order to produce a standardised, reproducible organotypic tissue, all lots of EpiDerm are compared to a reference database of effective time-50 (ET-50) values, i.e. the time of exposure after which viability is reduced to 50% following exposure to 100 microl of Triton X-100. The database average (184 tissue lots) is 6.74 +/- 0.99 hours (+/- 1 SD); initial lots of the full thickness tissue, tested in an identical manner, averaged 7.79 +/- 1.24 hours (n = 11). Histological cross-sections of the full thickness tissue showed an epidermal layer that is very similar to EpiDerm and native epidermis on a fibroblast-containing collagen matrix dermis-like layer. Irradiation of the tissue with ultraviolet light induced an increase in collagenase (MMP-1) release. Based on these initial results, investigation of the tissue response to stimuli specifically affecting the dermis or epidermal/dermal "cross-talk" will probably prove informative.

Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three-dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice.

To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP-13]) and collagenase 1 (MMP-1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3-dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice. Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell-cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts. Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3-dimensional collagen gels was associated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2-4-fold increase upon treatment with interleukin-1beta or tumor necrosis factor a. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3-dimensional collagen or by proinflammatory cytokines. The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3-dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell-matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.

Effects of genistein on the periovulatory expression of messenger ribonucleic acid for matrix metalloproteinases and tissue inhibitors of metalloproteinases in the rat ovary.

The matrix metalloproteinases (MMPs) play critical roles in the ovulatory process. Their expression and activity, together with those of the endogenous tissue inhibitors of metalloproteinases (TIMPs), are stimulated by LH. The LH surge initiates a cascade of events resulting in ovulation and formation of the corpus luteum via activation of protein kinases A and C, as well as tyrosine kinases. In vitro perfused rat ovaries were untreated, or treated with LH (0.2 microg ml(-1)) plus 0.2 mmol 3-isobutyl-1-methylxanthine l(-1) with 0, 10 or 100 micromol genistein l(-1) (an inhibitor of tyrosine kinases) to assess whether tyrosine kinases are mediators of the LH-stimulated increase in ovarian expression of the MMPs and TIMPs. After 10 h of perfusion, ovaries were collected and frozen until RNA isolation. Northern and RNase protection analyses were used to measure mRNA encoding collagenase 3, gelatinases A and B, and TIMPs-1, -2 and -3. Treatment with LH plus 3-isobutyl-1-methylxanthine resulted in a two- and fivefold increase in mRNA encoding collagenase 3 and TIMP-1, respectively (P < 0.05). Treatment with 100 micromol genistein l(-1) blocked the LH-stimulated increase in collagenase 3 (0.012 +/- 0.002 versus 0.028 +/- 0.005 relative units for 100 micromol genistein l(-1) versus LH; P < 0.05), whereas neither dose of genistein affected LH-induced TIMP-1 expression. LH alone or with genistein did not alter the expression of mRNA encoding TIMP-2 and TIMP-3, or mRNA encoding gelatinases A and B. These data indicate that tyrosine kinases play a role in the LH-induced tissue remodelling required for ovulation by mediating the LH-stimulated expression of collagenase 3.

Effects of genistein on the periovulatory expression of messenger ribonucleic acid for matrix metalloproteinases and tissue inhibitors of metalloproteinases in the rat ovary

The matrix metalloproteinases (MMPs) play critical roles in the ovulatory process. Their expression and activity, together with those of the endogenous tissue inhibitors of metalloproteinases (TIMPs), are stimulated by LH. The LH surge initiates a cascade of events resulting in ovulation and formation of the corpus luteum via activation of protein kinases A and C, as well as tyrosine kinases. In vitro perfused rat ovaries were untreated, or treated with LH (0.2 microg ml(-1)) plus 0.2 mmol 3-isobutyl-1-methylxanthine l(-1) with 0, 10 or 100 micromol genistein l(-1) (an inhibitor of tyrosine kinases) to assess whether tyrosine kinases are mediators of the LH-stimulated increase in ovarian expression of the MMPs and TIMPs. After 10 h of perfusion, ovaries were collected and frozen until RNA isolation. Northern and RNase protection analyses were used to measure mRNA encoding collagenase 3, gelatinases A and B, and TIMPs-1, -2 and -3. Treatment with LH plus 3-isobutyl-1-methylxanthine resulted in a two- and fivefold increase in mRNA encoding collagenase 3 and TIMP-1, respectively (P < 0.05). Treatment with 100 micromol genistein l(-1) blocked the LH-stimulated increase in collagenase 3 (0.012 +/- 0.002 versus 0.028 +/- 0.005 relative units for 100 micromol genistein l(-1) versus LH; P < 0.05), whereas neither dose of genistein affected LH-induced TIMP-1 expression. LH alone or with genistein did not alter the expression of mRNA encoding TIMP-2 and TIMP-3, or mRNA encoding gelatinases A and B. These data indicate that tyrosine kinases play a role in the LH-induced tissue remodelling required for ovulation by mediating the LH-stimulated expression of collagenase 3.

Suppression of T cell–mediated injury in human gut by interleukin 10: Role of matrix metalloproteinases

Activation of lamina propria T cells with pokeweed mitogen in human fetal gut explant cultures results in severe mucosal injury. In the same system, Staphylococcus aureus enterotoxin B produces villus atrophy and crypt cell hyperplasia. The aim of this study was to examine the ability of interleukin (IL)-10 to modify these changes. Mucosal pathology and lamina propria glycosaminoglycans were assessed histologically, and CD3(+), CD25(+) and alpha-actin smooth muscle-positive cells were determined by immunohistochemistry. Reverse plaque assays and quantitative reverse-transcriptase polymerase chain reaction were used to analyze the cytokine response. Matrix metalloproteinase production was analyzed by Western blotting, in situ hybridization, and zymography. Both experimental enteropathies produced mucosal damage, although injury was greater after pokeweed mitogen activation than S. aureus enterotoxin B. In both cases, the increase in cytokine-secreting cells and transcripts observed after T-cell activation was inhibited by IL-10. IL-10 also inhibited the increase in collagenase and stromelysin-1 in the explant culture supernatants and the loss of glycosaminoglycans. IL-10 decreased metalloproteinase production in control explants and increased matrix. In mesenchymal cells, IL-10 had a minor effect on metalloproteinase production. IL-10 down-regulates mucosal T-cell activation, metalloproteinase production, and loss of extracellular matrix and prevents mucosal damage in the gut.

Activation of Collagenase IV Gene Expression and Enzymatic Activity by the Moloney Murine Leukemia Virus Long Terminal Repeat

Moloney murine leukemia virus (Mo-MuLV) is a thymotropic and leukemogenic retrovirus which causes T lymphomas and leukemias, yet does not contain a transforming gene product. Mo-MuLV has been shown to trans-activate cellular genes via a polymerase III-generated transcript, designated let, from the long terminal repeat (LTR). Here we demonstrate that introduction of the Mo-MuLV LTR stably, or transiently, into murine or human cultured cells resulted in an 8- to 15-fold increase in collagenase IV (92-kDa gelatinase, gelatinase B, matrix metalloproteinase-9) gene expression. Collagenase IV protein expression was induced 9-fold by stable integration of MuLV LTR, as measured by immunoblot analysis using an anti-collagenase IV polyclonal antibody. The MuLV LTR coordinately stimulated the proteolytic activity of collagenase IV by 14-fold. The AP-1-binding site in the collagenase IV promoter was required for transactivation by the LTR. Collagenase type IV degrades type IV collagen, a major component of basement membrane, which constitutes the first step of the metastatic cascade. The activation of proteolytic enzymes by the MuLV LTR may thus play a contributory role in the development or spread of virus-induced lymphomas or leukemias.

Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals.

We have shown previously that the expression of collagenase is upregulated in rabbit synovial fibroblasts cultured on a substrate of antibody to the alpha 5 chain of the alpha 5 beta 1 integrin fibronectin receptor or on the 120-kD cell-binding chymotryptic fragment of plasma fibronectin, but remains at basal levels in cells plated on intact plasma fibronectin. We now have identified some of the components of a signaling pathway that couples the fibronectin receptor to the induction of collagenase transcription. We studied the control of collagenase gene expression in cells adhering to the 120-kD fragment of fibronectin, to antifibronectin receptor antibody, or to plasma fibronectin by transiently introducing promoter-reporter constructs into rabbit synovial fibroblasts before plating cells on these matrices. The constructs contained segments of the human collagenase promoter regulating transcription of chloramphenicol acyl transferase. Expression of constructs containing the -1200/-42-bp segment or the -139/-42-bp segment of the collagenase promoter inserted upstream from the reporter gene was induced to similar extents in cells plated on the 120-kD fragment of fibronectin or on anti-fibronectin receptor antibody, relative to that in fibroblasts plated on fibronectin. The expression of the construct containing the -66/-42-bp segment of the promoter was not regulated and was similar to that of the parent pBLCAT2 plasmid, suggesting that the -139/-67 region of the collagenase promoter, which contains PEA3- and AP1-binding sites, regulates the transcription of collagenase caused by integrin-derived signals. Expression of a reporter construct containing only the PEA3 and AP1 sites in the collagenase promoter (-90/-67) also increased in cells plated on the 120-kD fragment of fibronectin or on anti-fibronectin receptor antibody, relative to that in cells plated on fibronectin. Mutations in either the AP1 or PEA3 site of this minimal promoter abrogated its activity in cells plated on these inductive ligands. Expression of c-fos mRNA increased within 1 h of plating cells on the 120-kD fibronectin fragment or on anti-fibronectin receptor antibody, relative to that in cells plated on fibronectin. c-Fos protein accumulated in the nuclei of fibroblasts within 10 min of plating on the 120-kD fibronectin fragment. The increase in c-Fos was required for the increase in collagenase in cells plated on the 120-kD fibronectin fragment: incubation of cells with antisense, but not sense, c-fos oligonucleotides diminished both basal and induced expression of the -139/-42 collagenase promoter-reporter construct and decreased expression of the endogenous collagenase gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulatory effect of aminopeptidase inhibitor (bestatin) on the cervix during induction of ripening by interleukin-8

Bestatin is an immunomodulatory peptide that stimulates the humoral and cell-mediated immune system. It also has an inhibitory effect on multiple aminopeptidases. Recently we found that aminopeptidase N inactivates interleukin-8 in vitro. Bestatin successfully suppresses the effect of aminopeptidase N on interleukin-8. During cervical maturation many biochemical changes occur including decrease in collagen concentration and increase in collagenase and elastase activities. Interleukin-8, which has a potent neutrophil chemotactic effect, was found to induce cervical ripening in rabbits. The combination of interleukin-8 with bestatin also induced cervical ripening by providing approximately regular levels of neutrophil numbers, collagenase, and elastase activities. We therefore suggest that this regulatory mechanism also takes place in vivo through the inhibitory effect of bestatin on aminopeptidase N.

Expression of collagenase and stromelysin in skin fibroblasts from recessive dystrophic epidermolysis bullosa.

Collagenase and stromelysin expression in recessive dystrophic epidermolysis bullosa (RDEB) was studied at both the protein and the gene expression levels in fibroblast cultures. The amount of enzyme protein in the culture medium, as determined using a specific enzyme assay, showed a 9.7-fold increase in collagenase and a 2.7-fold increase in stromelysin in RDEB fibroblasts (n = 4 patients) compared with controls (n = 3 subjects with normal skin). Collagenase activity was extremely high in all RDEB fibroblasts. Gene expression, as assessed by Northern blot hybridization, was increased in two sets of RDEB fibroblasts with respect to collagenase, and in two other sets of RDEB fibroblasts with respect to stromelysin. The effect of interleukin-1 alpha (IL-1 alpha) on metalloproteinase expression was also examined. The results revealed that: 1) collagenase and stromelysin expression was variably increased at both the protein and the gene expression levels in RDEB fibroblasts; (2) the gene expression level did not always reflect the corresponding protein level; and (3) IL-1 alpha produced a differential effect on collagenase and stromelysin expression. Although the causative gene for RDEB is a type VII collagen, the abnormal expression of collagenase and/or stromelysin is still important in considering the pathophysiology of RDEB.

Serum collagenase levels during pregnancy and parturition

Recent studies have described the origin of the collagenase responsible for the degradation and loss of collagen during cervical ripening and dilatation to be polymorphonuclear leukocytes. In the present study the clinical feature of cervical dilatation was correlated to the serum levels of human granulocyte collagenase. The serum collagenase level was measured in 19 premenopausal women and in 181 pregnant women. In 15 of these pregnant women serum samples were obtained from the onset of labour to the active phase of labour at four different times. The collagenase concentrations remained low up to the 35th week of gestation (19.4 ng/ml) in comparison with the beginning of pregnancy. From the 35th week of gestation on we found a slight increase in collagenase in maternal serum. The maximum peak was reached during parturition at a cervical dilatation of 6-8 cm (71.2 ng/ml). The serum levels returned to non-pregnant values the first day following delivery. The results show that collagenases are critically involved in parturition.

Ultraviolet A Irradiation Stimulates Collagenase Production in Cultured Human Fibroblasts

This study was designed to investigate the biochemical mechanisms responsible for the connective tissue changes seen in actinically damaged skin, which is characterized histologically by diminution and ultrastructural alterations of collagen fibrils and deposition of elastotic material in the papillary dermis. We hypothesized that ultraviolet light could stimulate synthesis of interstitial collagenase in the skin, resulting in collagen degradation. Monolayer cultures of human fibroblasts or keratinocytes were irradiated with ultraviolet A (UVA) or ultraviolet B (UVB) radiation and interstitial collagenase or its inhibitor, TIMP (tissue inhibitor of metalloproteinases) assessed in the conditioned medium with Western immunoblots 24 h after irradiation. Northern blot analysis of the irradiated fibroblasts with a cDNA probe representing collagenase was also performed. Cell viability was greater than 90% with all doses of UV radiation studied. A dose-related increase in immunoreactive collagenase was detected in the medium of fibroblasts irradiated with 0-10 J/cm2 of UVA radiation as well as a parallel increase in the collagenase mRNA in the irradiated cells. UVA radiation stimulated collagenase synthesis in both neonatal and adult fibroblasts. TIMP production in UVA-irradiated fibroblasts increased to a lesser degree than did collagenase and its increase did not parallel the increase in collagenase. UVB (0-100 mJ/cm2) did not stimulate collagenase production by fibroblasts. In contrast to the stimulation of collagenase production by fibroblasts, a slight decrease in immunoreactive collagenase was seen in UVA-irradiated keratinocytes. These data suggest that direct stimulation of collagenase synthesis by human skin fibroblasts by UVA radiation may contribute to the connective tissue damage induced by ultraviolet radiation leading to photoaging.

Mechanism of mammalian ovulation

The sequence of events within the ovary during the process of ovulation discussed in this review is schematically represented in Fig. 1. It is obvious that LH, perhaps with some contribution from FSH, is the normal physiological trigger for the ovulatory sequence of events, and it appears from the available information that the effects of LH are mainly mediated via adenylate cyclase and increased cAMP levels. The cAMP in turn, via cAMP-dependent protein kinase, influences at least three distinct steps in the ovulatory process which seem to be of crucial importance, namely 1) the stimulation of steroidogenesis; 2) the stimulation of cyclooxygenase/lipooxygenase leading to increased prostaglandin/leukotriene synthesis; and 3) the stimulation of plasminogen activator which catalyzes the conversion of plasminogen to plasmin. A fourth crucial step in the ovulatory mechanism is the LH-induced increase in latent collagenase, but it remains to be determined if this step is mediated via cAMP. Concomitant with the increase in latent collagenase, there also appears to be an LH-dependent increase in collagenase inhibitors. The latent collagenase is then activated, and it appears that leukotrienes and prostaglandins, as well as plasmin, may be involved in this process. The active collagenase causes a digestion of the collagen in the follicle wall, and plasmin, as well as possibly other proteolytic enzymes such as proteoglycanases, may cause a further dissociation of the follicular wall. These processes of digestion of collagen and dissociation of the collagen fibers result in an opening in the follicular wall with the formation of the stigma and rupture. While the weakening of the follicular wall takes place throughout the entire wall, rupture remains for the most part a localized process at the apex of the follicle. This localization of the rupture may be explained on the basis of mechanical factors operating when the follicle wall thins and weakens. While it is clear that prostaglandins and leukotrienes can influence smooth muscle by causing contractions and that these compounds can cause vascular changes such as increased permeability, vasodilation, and vasoconstriction, it is not clear what the exact role of these latter processes are in ovulation. It appears that progesterone and not estrogen play an important role in the mechanism of LH-induced follicular rupture, but the locus of action of progesterone and its mechanism of action remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of Skin Fibroblast Collagenase Production by a Cytokine Derived from Basal Cell Carcinomas

Our previous studies of human basal cell carcinomas (BCC) revealed increased skin collagenase in vivo. Immunocytochemically the collagenase was localized to adjacent stroma, not to the tumor cells. When grown in culture, skin fibroblasts derived from tumor stroma showed a 3- to 4-fold increase in collagenase for the first 10-14 mean population doublings, after which collagenase expression reverted to control levels. These studies suggested that tumors stimulated adjacent fibroblasts to produce more collagenase. In the present study we sought direct evidence for epithelial-stromal interaction in this neoplasm. Under dissecting microscopy tumor islands were freed of stroma, homogenized, sonicated, and centrifuged to remove insoluble tissue. Tumor extracts were incubated with monolayer cultures of normal human skin fibroblasts to assess their effect on collagenase synthesis in these target cells. Culturing the fibroblasts for 24 h in the presence of individual BCC extracts resulted in a 1.6- to 3-fold increase in trypsin-activatable collagenase in the culture medium. This was paralleled by an equal increase in immunoreactive protein, suggesting enhanced enzyme synthesis. There was no change in the activity per immunoreactive protein, indicating a catalytically unaltered enzyme. Gel filtration of pooled BCC extracts showed that the stimulatory activity was contained in eluent fractions of Mr approximately 19Kd. These data suggest that BCCs elaborate a macromolecular cytokine that induces collagenase synthesis in skin fibroblasts and emphasize the importance of epithelial-stromal interactions in cutaneous tumor invasion.

Colchicine-Induced Modulation of Collagenase in Human Skin Fibroblast Cultures. II. A Probe for Defective Regulation in Epidermolysis Bullosa

The addition of colchicine to cultures of normal human skin fibroblasts produces a significant stimulation of collagenase. Because this finding implies a role for the microtubule system in the regulation of normal collagenase synthesis, we have used colchicine as a probe for aberrations in this enzyme in epidermolysis bullosa. In fibroblast cultures from the dominant simplex, dominant dystrophic, and recessive letalis forms of epidermolysis bullosa, 10(-6) M colchicine produced approximately a 2-fold increase in collagenase in the culture medium, a finding shown by biosynthetic studies to be attributable to enhanced synthesis of enzyme protein. In the case of typical recessive dystrophic epidermolysis bullosa, a disease characterized by excessive collagenase synthesis, the fibroblasts could also be stimulated to produce additional collagenase, despite having elevated baseline synthetic rates. In contrast, fibroblasts isolated from one recessive epidermolysis bullosa patient were resistant to the stimulatory effects of colchicine in concentrations up to 5 x 10(-6) M. In the absence of colchicine, collagenase synthesis in this patient's cells (termed REBc-) was 3-4 times that of normal controls, suggesting that the as yet undefined cellular function that is abrogated (or stimulated) by colchicine in normal cells may have been genetically impaired in these REBc- cells. Despite the resistance to colchicine, as manifested by the failure to stimulate collagenase, gross parameters of microtubular function, such as cell replication, were intact. Phenotypically, this patient had a form of epidermolysis bullosa intermediate between typical recessive dystrophic and recessive letalis forms of the disease. Although an experimentally induced blister was located in the lamina lucida, hypoplastic anchoring fibrils were also observed. These findings, in addition to the marked increase in collagenase synthesis, suggest the possibility that this patient may represent a compound heterozygote of two forms of epidermolysis bullosa and that colchicine may be useful in defining other such patients.

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6--8.2 than at pH 6.8--7.2. The pH-associated increase in collagenase was paralleled by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.

THE ROLE OF HUMAN SKIN COLLAGENASE IN EPIDERMOLYSIS BULLOSA

Human skin collagenase was quantitated by radioimmunoassay in 40 patients with various forms of epidermolysis bullosa to compare levels of the enzyme in blistered and clinically unaffected skin. Immunoreactive human skin collagenase was significantly elevated in the blistered skin of patients with both recessive and dominant forms of dystrophic epidermolysis bullosa (DEB). In addition, patients with generalized recessive DEB manifested a 4-fold increase in collagenase protein in normal-appearing skin, and patients with localized recessive DEB or epidermolysis bullosa letalis showed a 3-t to 3.5-fold elevation in the enzyme. However, patients with dominantly inherited DEB failed to displays a statistically significant increase in immunoreactive collagenase in nonblistered skin. Although it cannot be definitely stated whether the elevated collagenase content in the blistered skin represents a primary or secondary event, such as part of a wound healing response, the demonstration of markedly increased levels of collagenase in normal-appearing skin could, in part, provide an explanation at the molecular level for the formation of blisters in this disease.