βig-h3 supports keratinocyte adhesion, migration, and proliferation through α3β1 integrin

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Abstract

βig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-β and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that βig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-β in normal human keratinocytes (NHEK) and HaCaT cells. βig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with α3β1 integrin. Previously identified two α3β1 integrin-interacting motifs of βig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that βig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.

Section snippets

Materials and methods

Immunohistochemistry. Normal human skin was obtained during plastic surgery and embedded in paraffin. Five-micrometer sections were placed onto slide, deparaffinized, and rehydrated. Sections were incubated in blocking solution (1% BSA, 0.2% gelatine, and 0.05% saponin in PBS) for 30 min at room temperature, followed by incubation with anti-human βig-h3 antibody overnight at 4 °C. The sections were washed and incubated with peroxidase-conjugated anti-rabbit IgG antibody (DAKO, Glostrup, Denmark)

βig-h3 is present in the papillary dermis and synthesized by basal keratinocytes

Histological sections were examined to localize βig-h3 expression in normal skin. Immunohistochemical staining showed that a positive staining was observed in a band along the upper dermal layer (Figs. 1A and B). Control staining using rabbit IgG was negative (data not shown). Unlike the results of LeBaron et al. [6], we could not detect any staining in the granular layer of epidermis. Instead, we had a positive signal in the basal keratinocytes by in situ hybridization (Figs. 1C and D). The

Discussion

βig-h3 was first identified as a gene induced in A549 cells after treatment with TGF-β [3] and subsequently reported to be induced by TGF-β in several cell types. It has been reported to exist in several tissues [4], [5], [6], [7], [8]. Although biological roles of βig-h3 are largely unknown, it has been suggested that it may act as a cell adhesion substrate, regulate cell growth, interconnect other matrix components, and transduce TGF-β-mediated signaling [3], [4], [6], [9]. The results

Acknowledgements

This work was supported by a program of National Research Laboratory (M10104000036-01J0000-01610).

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    Abbreviations: fas-1, fasciclin 1 homologous domain; HCE, human corneal epithelium; ECM, extracellular matrix; pFN, plasma fibronectin; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FBS, fetal bovine serum.

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