Abstract
We previously found that a rat CLCA homologue (rCLCA-f) modulates Ca(2+)-dependent Cl(-) transport in the ductal cells of the rat submandibular gland. CLCA proteins have been shown to be multifunctional, with roles in, for example, cell adhesion. Here, we describe the mRNA and protein expressions of a splicing isoform of rat rCLCA (rCLCA-t). This isoform is a 514-amino acid protein containing a C-terminal 59-amino acid that is distinct from the rCLCA-f sequence. Immunohistochemistry revealed rCLCA-t to be located in the basal cells of the rat submandibular gland excretory duct and the stratum basale of rat epidermis, whereas rCLCA-f was detected in cells during the process of differentiation. In a heterologous expression system, rCLCA-t was found to be a membrane protein present predominantly in the perinuclear region, and not to be either present on the cell surface or secreted. rCLCA-t failed to enhance ionomycin-induced Cl(-) conductance (unlike rCLCA-f). When compared with rCLCA-f, it weakened cell attachment to a greater extent and in a manner that was evidently modulated by intracellular Ca(2+), protein kinase C, and β(1)-integrin. rCLCA-t was found to associate with RACK1 (receptor for activated C kinase) and to reduce expression of mature β(1)-integrin. Treatment of rat skin with rCLCA-t siRNA increased the expression of β(1)-integrin in the stratum basale of the epidermis. These results are consistent with cell-specific splicing of rCLCA mRNA playing a role in the modulation of the adhesive potential of undifferentiated epithelial cells.
Highlights
CLCA family members have been shown to be multifunctional proteins
Immunohistochemistry revealed rat CLCA homologue (rCLCA)-t to be located in the basal cells of the rat submandibular gland excretory duct and the stratum basale of rat epidermis, whereas rCLCA-f was detected in cells during the process of differentiation
When compared with rCLCA-f, it weakened cell attachment to a greater extent and in a manner that was modulated by intracellular Ca2؉, protein kinase C, and 1-integrin. rCLCA-t was found to associate with RACK1 and to reduce expression of mature 1-integrin
Summary
CLCA family members have been shown to be multifunctional proteins. Results: A rat splicing isoform was localized to the stratum basale of the epidermis. Treatment of rat skin with rCLCA-t siRNA increased the expression of 1-integrin in the stratum basale of the epidermis These results are consistent with cell-specific splicing of rCLCA mRNA playing a role in the modulation of the adhesive potential of undifferentiated epithelial cells. The present study suggests (a) that heterologous rCLCA-t expression may cause an attenuation of 1-integrin-mediated cell adhesion, with no alteration in ClϪ conductance, in human embryonic kidney 293 (HEK293) cells, and (b) that isoform-specific knockdown increases 1-integrin expression in the basal layer of the skin These results lead us to hypothesize that this cell-specific CLCA isoform might help to confer novel functions, such as modulation of adhesive potential, on undifferentiated epithelium
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