L. A. DeFreest1, G. J. DeFreest-Rondeau1, L. Van De Water1, C. M. DiPersio1 1Albany Medical College,Department Of Surgery,Albany, NY, USA
Introduction: Integrins expressed in epidermal keratinocytes have important roles in cutaneous wound healing including paracrine stimulation of angiogenesis, regulation of proliferation and migration, and remodeling of extracellular matrix (ECM). Our laboratory has investigated roles of the laminin-binding integrin α3β1 in keratinocytes using both in vivo and in vitro models of wound healing. Our previous studies support a critical role for α3β1 in regulating the secretion of proteins into the extracellular milieu. Current data indicate a gene regulatory mechanism that involves crosstalk between α3β1 and estrogen responsive pathways in keratinocytes. Our long-term goal is to elucidate how α3β1 and estrogen coordinately regulate paracrine and autocrine signaling mechanisms that modulate the wound microenvironment to promote efficient wound healing.
Methods: For these studies, we have established a cell culture model consisting of mouse keratinocytes (MK cells), that are homozygous for a null mutation in the gene that encodes the α3 integrin subunit (i.e. lacking α3β1), or MK cells stably transfected with human α3 (i.e. MK α3+ cells, expressing α3β1). Conditioned media collected from these cells were compared by mass spectrometry (MS) to identify proteins secreted in an α3β1–dependent manner. Candidate proteins were then further evaluated by immunoblot to confirm and quantitate their expression/secretion. MK cells were also evaluated for the presence of estrogen receptor (ER) isoforms.
Results: 19 proteins identified by MS were upregulated more than 4-fold in medium from MK α3+ cells, compared with MK α3– cells. The secretion of lactoferrin, a protein whose production is controlled through an ER-dependent pathway, was up-regulated 18-fold in MK α3+ cells which was confirmed by immunoblot. Preliminary results indicate that MK cells express both ERα and ERβ. Furthermore, ERβ was increased in MK α3+ cells compared with MK α3- cells while ERα levels were similar in both cell lines. ERβ and ERα were increased in both MK lines by treatment with estrogen for 24 hours.
Conclusion: Proteomic analysis identified numerous secreted proteins that are upregulated by α3β1 indicating an important role for this integrin in controlling the keratinocyte secretome. Among these proteins, lactoferrin has been shown to modulate wound healing and has anti-microbial and immunomodulatory properties that make it an interesting candidate for further study. Lactoferrin is known to be induced by estrogen signaling, suggesting an unexplored intersection of pathways governed by integrins and ER. In particular, regulation of ERβ levels downstream of α3β1 may be a novel intracellular signaling pathway to regulate production of paracrine factors which in turn, modulate the wound microenvironment.