Expression Profiling of TRPC Channels in Human Gingival Keratinocytes

Objective: To determine whether hTRPC channels are expressed in proliferating and/or differentiating human gingival keratinocytes (HGKs). Methods: Calcium is an important regulator of keratinocyte differentiation in vitro. Hence, HGKs were cultured in 0.03 mM external [Ca2+] to keep them in a proliferative and undifferentiated state (Infect. Immun. 63:3878, 1995); and cultured in 1.2 mM external [Ca2+] to induce them to cease proliferating and undergo differentiation. Involucrin expression was used to determine differentiation status. hTRPC channel mRNAs were detected by RT-PCR in total RNA extracts from nonconfluent and confluent cell cultures. Western blot analysis of total protein or membrane protein extracts (J. Biol. Chem. 277:34462, 2002) and immunohistochemistry were used to evaluate protein expression. Antibodies were only available for TRPC1 and TRPC6 (Alomone Labs, Ltd.). Results: TRPC1, TRPC5, TRPC6 and TRPC7 mRNAs were detected in proliferating cultured keratinocytes. (TRPC4 was also present, and its results are reported separately.) Immunohistochemistry demonstrated that TRPC1 and TRPC6 were expressed in gingival tissue in basal and suprabasal epithelial layers. mRNA levels for the first three channels in cultured cells increased for 8-96 hours after switching to 1.2 mM external [Ca2+], then decreased over 4-8 days. TRPC1 and TRPC6 protein levels were stable to 8 days. Involucrin expression was low in proliferating cells and markedly increased at 2-8 days. Conclusion: TRPC channel mRNAs were decreased in differentiated HGKs (i.e., in cells with high involucrin levels), but channel protein expression was stable. This indicates TRPC channels may be important in Ca2+ signaling in proliferating and in differentiating keratinocytes. Supported by NIH grants AR 46254 and DE 11111.