Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Termination of wound healing requires a fine balance between collagen deposition and its hydrolysis. To dissect the underlying control mechanisms for this process, we established a keratinocyte/fibroblast co-culture system and subsequently demonstrated more than a 10-fold increase in collagenase expression in fibroblasts co-cultured with keratinocytes relative to that of control cells. This finding was further confirmed in fibroblasts grown in a keratinocyte/fibroblast collagen-GAG gel. The efficacy of keratinocyte-derived collagenase stimulatory factors on collagenase activity was evaluated, and the results showed that only conditioned medium derived from fibroblasts co-cultured with keratinocytes was able to break down markedly type I collagen to its one-quarter and three-quarter fragments of both alpha (alpha1 and alpha2) and beta (beta1.1 and beta1.2) chains. The results of a dose-response experiment showed that keratinocyte-conditioned medium (KCM) stimulates the expression of collagenase mRNA by dermal fibroblasts in a concentration-dependent fashion. In a similar experiment, the results of a time-response experiment revealed that KCM treatment increases the expression of collagenase mRNA in dermal fibroblasts as early as 6 h and reaches its maximum level within 24-48 h. Considering that this keratinocyte-releasable factor has a potent collagenase stimulatory effect on fibroblasts, which favors the resolution of accumulated type I and type III collagen found in fibrotic tissue, we referred to this protein as a keratinocyte-derived anti-fibrogenic factor (KDAF). In a series of chromatography experiments and a direct trypsin digestion of the proteins and subsequent peptide mapping, a keratinocyte-derived collagenase-stimulating factor turned out to be a releasable form of stratifin, also known as 14-3-3 sigma protein. To validate this finding, stratifin cDNA was cloned into a pGEX-6P-1 expressing vector and more than 50 mg of recombinant stratifin was generated and used to treat fibroblasts with various concentrations for 24 h. The results of northern analysis showed a remarkable dose-response increase in the expression of collagenase mRNA in stratifin-treated fibroblasts relative to that of the control. This finding was consistent with that obtained from collagenase activity assay. In conclusion, we identified a keratinocyte-releasable form of stratifin in KCM that mimics the collagenase stimulatory effect of KCM for dermal fibroblasts. This finding suggests that stratifin is likely to be, at least, one of the KDAFs found in KCM.
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PMID:Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts. 1514 Feb 22

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.
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PMID:Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts. 1564 39

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.
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PMID:Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts. 1565 40

Previously, we have demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, stimulates matrix metalloproteinase (MMP)-1 expression in dermal fibroblasts. In this study, we showed that stratifin induced fibroblast MMP-1 messenger ribonucleic acid (mRNA) and protein levels through p38 mitogen-activated protein kinase (MAPK). Our data indicated that treatment of dermal fibroblasts with stratifin resulted in rapid and transient upregulation of c-jun and c-fos mRNA levels. We also demonstrated that SB203580 (SB), a specific inhibitor of p38 MAPK activity, inhibited the activation of fibroblast MMP-1 mRNA expression by stratifin. Subsequently, western blot analysis revealed phosphorylation of p38 at 90 min after stratifin stimulation and this was decreased to approximately 50% of the maximum value by 120 min. Stratifin was demonstrated to increase MMP-1 protein levels starting at 4 h and reaching its peak at 12-24 h. Furthermore, SB significantly blocked the stratifin induction of MMP-1 protein levels (***p<0.005, n=3). Microarray analysis of stratifin-treated fibroblasts shows an increase in Elk4/Sap1 mRNA expression and this finding was confirmed by northern blot analysis. Our results indicate that stratifin markedly increase Elk4/Sap1 mRNA expression in a time-dependent fashion. In conclusion, stratifin stimulates fibroblast MMP-1 levels through the activation of c-fos and MAPK pathway.
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PMID:Stratifin-induced matrix metalloproteinase-1 in fibroblast is mediated by c-fos and p38 mitogen-activated protein kinase activation. 1609 31

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions.
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PMID:Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin. 1644 Mar 5

Corneal angiogenesis is associated with a variety of corneal diseases, and is sometimes vision threatening. In recent years, with the discovery of major pro- and anti-angiogenic factors in the cornea, details of the angiogenic process are gradually unveiled. Of note, corneal inflammation and neovascularization associated with severe limbal stem cell (LSC) deficiency is a clinically challenging issue in that the condition persists long after the initial insult, and will not improve without transplantation of LSCs. However, to date the molecular mechanism by which LSC transplantation restores corneal avascularity is not fully understood. In addition to discussing major pro-angiogenic factors involved in corneal neovascularization, this review article also focuses on possible molecular mechanisms underlying persistent inflammation and neovascularization following severe LSC deficiency, and anti-angiogenic factors expressed by human limbo-corneal epithelial cells (HLCECs). Most of the recently discovered corneal anti-angiogenic factors belong to extracellular matrix proteins that acquire angio-inhibitory activity only after proper proteolytic processing. Our recent findings showed that the secretion of endostatin (derived from basement membrane collagen XVIII) and restin (from collagen XV) by HLCECs were enhanced when HLCECs were cultivated on amniotic membrane (AM). This adds to the advantage of transplanting ex vivo expanded HLCECs cultivated on AM in that the anti-angiogenic activity of the epithelial cells is augmented in a physiological way. Furthermore, proteomic profiling of HLCECs and human conjunctival epithelial cells (HCECs) identified a 14-3-3 protein (stratifin) preferentially expressed by HLCECs. In addition to functioning as a cell cycle controller, keratinocyte-derived stratifin induces MMPs which are involved in the generation of restin (by MMP-1) and endostatin (by MMP-3). These findings highlight the significance of delicate epithelial-matrix interactions in the maintenance of corneal avascularity.
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PMID:Regulation of corneal angiogenesis in limbal stem cell deficiency. 1707 82

Overexpression of wound healing-promoting factors such as transforming growth factor-1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) during the healing process has been implicated in the development of dermal fibrosis in patients following thermal injury, surgical incision, and deep trauma. However, the mechanism through which the expression of these two fibrogenic factors is slowed down and/or abrogated in the late stages of the healing process is not known. Here, we hypothesize that keratinocyte-releasable factors counteract the fibrogenic role of both IGF-1 and TGF-beta1 in fibroblasts. To test this hypothesis, the levels of collagenase (MMP-1), as an index for extracellular matrix degradation, in dermal fibroblasts in response to either keratinocyte-conditioned medium (KCM) or our recently identified keratinocyte-releasable stratifin in the presence and absence of either IGF-1, TGF-beta1, or both were evaluated. The results of Northern analysis showed a significant increase in collagenase mRNA expression in cells treated with KCM in the presence of both IGF-1 and TGF-beta1. The effect was, at least in part, due to keratinocyte-derived stratifin that was present in KCM. This was ascertained as the levels of MMP-1 mRNA were markedly reduced when cells were treated with stratifin-immuno-depleted KCM. The results of Western blot analysis showed an increase in the level of MMP-1 protein in stratifin-treated fibroblasts and this was consistent with the level of MMP-1 mRNA expression detected by Northern analysis. However, in contrast to KCM, whose efficacy on MMP-1 expression was modestly reduced by either IGF-1 and TGF-beta1, or a combination of both, these factors abrogated the MMP-1 stimulatory effect of stratifin in fibroblasts. In summary, the results of this study revealed that both stratifin and KCM stimulate the expression of MMP-1-in fibroblasts and this effect can be abrogated by either IGF-1, TGF-beta1, or a combination of both.
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PMID:Keratinocyte conditioned medium abrogates the modulatory effects of IGF-1 and TGF-beta1 on collagenase expression in dermal fibroblasts. 1735 56

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a "stemness condition" of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.
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PMID:The role of stratifin in fibroblast-keratinocyte interaction. 1764 30

Although, stratifin (SFN) is externalized by keratinocytes and stimulates the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts, its mechanism of externalization is not known. Here, we hypothesize that keratinocytes have a capacity to release stratifin through externalization of exosomes. To test this hypothesis, exosomes were purified from human keratinocyte conditioned medium (KCM) and analyzed for the presence of SFN by Western blot analysis using lysosomal-associated membrane protein 2 (LAMP-2) and heat shock cognate 70 (hsc70) as exosomal markers. The results showed the presence of SFN in keratinocyte lysate, concentrated KCM and exosomes, but not in concentrated unconditioned medium. Transmission electron microscopic examination revealed the presence of unique "saucer-like" structures characteristic of exosomes whose diameters were <100 nm. Similar to the recombinant SFN, the exosomes associated proteins stimulated MMP-1 expression in fibroblasts. Depletion of the exosomes markedly reduced this MMP-1 stimulatory effect. To further statistically confirm these findings, fibroblasts were treated with three different exosome preparations and the finding showed more than 7.4-fold increase in the level of MMP-1 in the treated cells. Furthermore, we found that approximately 1% of the total proteins contained in exosomes correspond to SFN. In conclusion, this study is the first report showing that keratinocytes have the capacity to produce exosomes through which some intracellular proteins such as SFN, with MMP-1 stimulating activity for fibroblasts, is externalized into keratinocyte microenvironment.
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PMID:Primary human keratinocytes externalize stratifin protein via exosomes. 1845 39

Transdifferentiation is a process in which the original commitment of a cell is changed to give rise to unexpected peripheral mature cells. Our previous report showed that circulating stem cells can generate keratinocyte-like cells (KLCs). However, it remains to be determined whether or not other peripheral blood mononuclear cells (PBMC) subsets have the potential to follow the same cell fate. In this study, the cell transdifferentiation of circulating CD14(+) monocytes into KLCs and their regulatory effect on matrix metalloproteinase-1 (MMP-1) expression in dermal fibroblasts were evaluated. The results showed that monocytes isolated from peripheral blood mononuclear cells have the capacity to generate KLCs. These transdifferentiated cells exhibited, along with a keratinocyte-like morphology, a characteristic profile consisting in stratifin(+), cytokeratins(+) (types I and II), CD14(low), and involucrin(+) on day 21 in culture. Similar to keratinocyte-conditioned media, KLC-derived conditioned media were able to induce an increase in the MMP-1 expression in dermal fibroblasts. This effect was significantly reduced by using 14-3-3 protein-depleted KLC-conditioned media. Our findings show the potential transdifferentiation of circulating CD14(+) monocytes into KLCs and their regulatory effect on MMP-1 expression in dermal fibroblasts.
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PMID:Circulating monocytes have the capacity to be transdifferentiated into keratinocyte-like cells. 1932 Aug 96


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