Epidermal basement membrane forms anchoring complex composed of hemidesmosomes, anchoring filaments, lamina densa and anchoring fibrils to link epidermis to dermis. However, the anchoring complex is rarely formed in skin equivalent models, probably because of degradation of extracellular matrix (ECM) proteins and heparan sulfate chains by matrix metalloproteinases (MMPs) and heparanase, respectively. To explore the roles of ECM proteins and heparan sulfate in anchoring complex assembly, we used specific inhibitors of MMPs and heparanase, and the formation of anchoring complex was analysed in terms of polarized deposition of collagen VII, BP180 and β4 integrin at the dermal-epidermal junction (DEJ) by means of immunohistochemistry and transmission electron microscopy (TEM). The deposition of collagen VII was polarized to the basal side by the addition of MMP inhibitor, and the staining intensity was increased by combined treatment with MMP inhibitor and heparanase inhibitor, which enhanced anchoring fibril formation as observed by TEM. BP180 was polarized to the basal side by heparanase inhibitor, which protects HS chains, but not by MMP inhibitor. MMP inhibitor improved the polarization of β4 integrin. Hemidesmosomes were formed in the presence of each inhibitor, as observed by TEM, and formation was greatly enhanced by the combined treatment. These findings suggest that heparan sulfate chains, in addition to ECM proteins at the DEJ, play an important role in the assembly of anchoring complex, especially hemidesmosomes and anchoring fibrils.
© 2011 John Wiley & Sons A/S.