Wound healing potential of a dimeric InlB variant analyzed by in vitro experiments on re-epithelialization of human skin models.

A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.

[1]  Manidipa Banerjee,et al.  GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation , 2004, Molecular microbiology.

[2]  G. Winter,et al.  Formation of the Scab and the Rate of Epithelization of Superficial Wounds in the Skin of the Young Domestic Pig , 1962, Nature.

[3]  W. Birchmeier,et al.  Met, metastasis, motility and more , 2003, Nature Reviews Molecular Cell Biology.

[4]  H. Tsubouchi,et al.  Phorbol ester‐induced secretion of human hepatocyte growth factor by human skin fibroblasts and its inhibition by dexamethasone , 1992, FEBS letters.

[5]  C. Liang,et al.  In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro , 2007, Nature Protocols.

[6]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[7]  N. Fusenig,et al.  Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones. , 1989, Differentiation; research in biological diversity.

[8]  M. Skobe,et al.  Platelet-derived growth factor-BB controls epithelial tumor phenotype by differential growth factor regulation in stromal cells. , 2006, The American journal of pathology.

[9]  M. Gossen,et al.  Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence‐activated cell sorting , 2010, Protein science : a publication of the Protein Society.

[10]  P. Cossart,et al.  Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family , 1995, Molecular microbiology.

[11]  F. Watt,et al.  Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes. , 1987, Experimental cell research.

[12]  E. Gherardi,et al.  Ligand-mediated dimerization of the Met receptor tyrosine kinase by the bacterial invasion protein InlB. , 2010, Journal of molecular biology.

[13]  M. Naujokas,et al.  InlB-Dependent Internalization of Listeria Is Mediated by the Met Receptor Tyrosine Kinase , 2000, Cell.

[14]  H. Niemann,et al.  Fold and Function of the InlB B-repeat* , 2011, The Journal of Biological Chemistry.

[15]  E. Gherardi,et al.  Structure of the Human Receptor Tyrosine Kinase Met in Complex with the Listeria Invasion Protein InlB , 2007, Cell.

[16]  J. Hornung,et al.  Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line , 1988, The Journal of cell biology.

[17]  S. Werner,et al.  c-Met is essential for wound healing in the skin , 2007, The Journal of cell biology.

[18]  S. Mizuno,et al.  HGF–MET Cascade, a Key Target for Inhibiting Cancer Metastasis: The Impact of NK4 Discovery on Cancer Biology and Therapeutics , 2013, International journal of molecular sciences.

[19]  T. Sun,et al.  Differentiation of the epidermal keratinocyte in cell culture: Formation of the cornified envelope , 1976, Cell.

[20]  M. Paulsson,et al.  Alternative proteolytic processing of hepatocyte growth factor during wound repair. , 2009, The American journal of pathology.

[21]  K. Matsumoto,et al.  Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. , 1991, Experimental cell research.

[22]  Nikolaos A. Peppas,et al.  PREPARATION, STRUCTURE AND DIFFUSIONAL BEHAVIOR OF HYDROGELS IN CONTROLLED RELEASE , 1993 .

[23]  H. Zwick,et al.  Unfixed material embedded in a methacrylate resin (Technovit 7100) for immunofluorescent staining. , 1987, Stain technology.