Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.
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.
Ghaffari
Abdi
A
Department of Surgery, BC Professional Firefighter's Burn and Wound Healing Research Lab, University of British Columbia, Vancouver, Canada.
Li
Yunyaun
Y
Karami
Ali
A
Ghaffari
Mazyar
M
Tredget
Edward E
EE
Ghahary
Aziz
A
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D024042:Collagen Type I / Q000096:biosynthesis; D017364:Collagenases / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D020405:Dermis / Q000166:cytology; D005092:Exonucleases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005095:Exoribonucleases; D005109:Extracellular Matrix / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D019714:Tissue Inhibitor of Metalloproteinases; D014945:Wound Healing / Q000502:physiology
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D024042:Collagen Type I; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D019714:Tissue Inhibitor of Metalloproteinases; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human; D017364:Collagenases; D020782:Matrix Metalloproteinases; C417249:collagenase 1; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1