Protein Kinase C Isoforms in Human and Rat Colonic Mucosa

Abstract

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC α, β, δ, ϵ, and ζ were detected in both rat and human colonic mucosa, while PKC η was detected in human colonic mucosa only. PKC α, β, and ζ were predominantly localized in the cytosolic fraction, whereas the majority of PKC δ, ϵ, and η were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucose, mRNA for PKC α, β, δ, ϵ, η and ζ were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC β, δ, ϵ, η and ζ. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucose expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.