L. A. DeFreest1, L. Van De Water2, C. DiPersio2 1Albany Medical College,Department Of Surgery, Division Of General Surgery, Section Of Trauma And Critical Care,Albany, NY, USA 2Albany Medical College,Department Of Surgery And Department Of Regenerative & Cancer Cell Biology,Albany, NY, USA
Introduction: Integrin receptors expressed in epidermal keratinocytes have been shown to have important roles in cutaneous wound healing including paracrine stimulation of angiogenesis, regulation of keratinocyte proliferation and migration, and production and remodeling of the extracellular matrix (ECM) to which they adhere. Our laboratory has investigated the roles of the laminin-binding integrin α3β1 in keratinocytes using both in vivo and in vitro models of wound healing. Our previous data supports a role for α3β1 in modulation of these processes by regulating the secretion of proteins into the extracellular milieu. Our long term goal is to elucidate the α3β1-dependent paracrine and autocrine signaling mechanisms that modulate the wound microenvironment to promote efficient wound healing.
Methods: Using a cell culture model of activated mouse keratinocytes (MK cells), we have prepared medium conditioned by MK cells that are homozygous for a null mutation in the gene that encodes the α3 integrin subunit (i.e. lacking α3β1 integrin), or MK cells stably transfected with human α3 (i.e. expressing α3β1 integrin). These conditioned media were analyzed by mass spectrometry to identify proteins secreted by MK cells in an α3β1–dependent manner, with a focus on those proteins that were upregulated at least four-fold in the presence of α3β1. Selected proteins in both media and lysates from MK cells were then evaluated by western blot to confirm and quantitate the secreted proteins.
Results: Of the >7000 proteins identified by mass spectrometry, nineteen were upregulated more than four-fold in medium from α3β1–expressing mouse keratinocytes. These proteins corresponded to a wide variety of keratinocyte functions including ECM, pro-angiogenic, and immunological species. Interestingly, lactotransferrin (LTfn) secretion was up-regulated 18-fold in α3β1-expressing cells. Western blot confirmed a significant upregulation of LTfn secretion as well as production of the protein in cell lysates.
Conclusion: Proteomic analysis of conditioned media from MK cell lines identified numerous secreted proteins that are upregulated by α3β1 indicating an important role for this integrin in controlling the keratinocyte secretome. Lactotransferrin has previously been shown to have a role in wound healing and has anti-microbial and immunomodulatory properties making it an interesting candidate for further study. Interestingly, LTfn is known to be expressed in response to estrogen and under the control of the estrogen receptor, suggesting an intersection of signaling pathways downstream of integrins and the estrogen receptor which has not been previously studied in detail. Elucidation of the signaling mechanisms that result in α3β1–dependent production of LTfn may lead to novel therapeutics to promote closure of surgical wounds.