Mitchel Villereal, Ph.D.

Signal Transduction Pathways in Normal and Transformed Cells

Research Summary

Signal transduction pathways in normal and transformed cells
My laboratory utilizes biochemical, biophysical, molecular, and proteomic approaches to study cell signaling pathways. We use human fibroblasts grown in cell culture as one model cell system to investigate early signaling events that are initiated when cells are stimulated with mitogens. We also use HEK-293 cells for studies where we introduce exogenous genes or siRNA constructs against endogenous genes, or, in some cases, fibroblasts derived from transgenic mice to evaluate the role of selected proteins in cell growth control. We also work in a cultured hippocampal cell system (H19-7 cells) when investigating questions pertaining to excitable cells. There are basically two areas of interest in our laboratory: 1) the mechanism for regulation of intracellular Ca2+ in response to mitogen stimulation and the downstream events regulated by Ca2+ entry and 2) the use of proteomics to elucidate tyrosine phosphorylation pathways downstream of the bradykinin (BK) receptor.

For the project on Ca2+ regulation, we are utilizing Ca2+-sensitive fluorescence indicators combined with sophisticated image analysis techniques to monitor mitogen-induced Ca2+ changes in cultured cells. We are particularly interested in the mechanism for stimulation of Ca2+ entry via either receptor-operated channels or store-operated channels. The latter are plasma membrane channels whose activity are stimulated in response to depletion of intracellular Ca2+ stores, a process that seems to utilize unique signaling pathways. We have recently provided evidence for the involvement of tyrosine kinase activity in the regulation of Ca2+ entry via store-operated channels. We have demonstrated a role for c-src in the regulation of Ca2+ entry based on studies utilizing transfection techniques to overexpress c-src, as well as fibroblasts derived from c-src knockout transgenic mice. We are utilizing biochemical and molecular approaches to identify which targets of c-src are important in the regulation of store-operated channels.

On another front, we are trying to identify the proteins responsible for forming the store-operated and receptor-operated Ca2+ channels. We used RT-PCR methods to screen for expression of mammalian homologs of Drosophila Trp (the gene hypothesized to code for store-operated Ca2+ channels in Drosophila) in HEK-293 or H19-7 cell. These cells express up to six of the 7 TRPC (a subfamily of Trp genes) proteins identified. To date, we have made constructs which express hairpin siRNA specific for individual TRPC homologs and we stably express these constructs in HEK-293 or H19-7 cells to selectively suppress one or more TRPC homologs to evaluate the role of these proteins in both store-operated and carbachol-stimulated Ca2+ entry. We have demonstrated that TRPC1 and TRPC3 are involved in mediating store-operated Ca2+ entry in both cell types, and TRPC7 is involved in HEK-293 cells but not in H19-7 cells. On the other hand, TRPC4 plays no role in store-operated Ca2+ entry in either cell type, but plays a major role in mediating carbachol-stimulated Ca2+ entry in HEK-293 cells. Of particular interest is our observation that in cells where expression of TRPC4 is selectively suppressed, low doses of carbachol can no longer generate repetitive Ca2+ oscillations. This indicates that TRPC4 mediates the Ca2+ entry required to maintain continuous Ca2+ oscillations in response to carbachol. We are continuing to analyze the contribution of TRPC5 and TRPC6, and combinations of various TRPC homologs, to Ca2+ channel activity initiated by a variety of stimuli (EGF, UV radiation, apoptosis stimuli, and cell cycle variations). We also will investigate the role of Ca2+ entry, via various TRPC channels, on downstream events such as transcription, cell growth, and apoptosis. We have recently identified a peptide toxin from scorpion venom that selectively inhibits store-operated Ca2+ channels and this toxin will be useful in identifying events regulated downstream of store-operated Ca2+ channels.

For the project on BK-induced tyrosine phosphorylation, we are stimulating HEK-293 cells expressing the B2 BK receptor with bradykinin, extracting cell proteins, purifying tyrosine phosphorylated proteins on an immunoaffinity column, and using the fraction specifically eluted from the column to identify the tyrosine phosphorylated proteins using a proteomics approach. We run the purified proteins on 2D gels, cut out protein spots that are differentially regulated by BK, and do peptide mass fingerprinting by mass spectroscopy to identify the proteins of interest. These studies are done in collaboration with Argonne National Laboratory.

Selected Papers

Owen NE and Villereal ML. (1983). Bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts. Cell, 32, 979-985.

Byron KL, Babnigg G and Villereal ML. (1992). Bradykinin-induced Ca entry, release and refilling of intracellular Ca+2 stores: Relationships revealed by image analysis of individual human fibroblasts. J. Biol. Chem., 267, 108-118.

Baumgarten LB, Toscas K and Villereal ML. (1992). Dihydropyridine-sensitive L-type Ca2+ channels in human fibroblast cells: Characterization of activation with the growth factor lys-bradykinin. J. Biol. Chem., 267, 10524-10530.

Lee KM, Toscas K and Villereal ML. (1993). Inhibition of bradykinin and thapsigargin-induced Ca2+ entry by tyrosine kinase inhibitors. J. Biol. Chem., 268, 9945-9948.

McSwine R, Babnigg G, Musch M, Chang E and Villereal ML. (1994). Expression and phosphorylation of NHE1 in wild-type and transformed human and rodent fibroblasts. J. Cell. Physiol., 161, 351-357.

Lee KL and Villereal ML. (1996). Tyrosine phosphorylation and activation of tyrosine kinases, pp60c-src and pp125FAK in human fibroblasts stimulated by bradykinin. Am. J. Physiol., 39, C1430-C1437.

McSwine R, Li J and Villereal ML. (1996). Examination of the role for Ca2+ in regulation and phosphorylation of the Na+/H+ antiporter NHE1 via mitogen and hypertonic stimulation. J. Cell. Physiol., 168, 8-17.

Babnigg G, Bowersox SR and Villereal ML. (1997). The role of pp60c-src in the regulation of calcium entry via store-operated calcium channels. J. Biol.Chem., 272, 29434-29437.

Babnigg G, Heller B and Villereal ML. (2000). Cell-to-cell variation in store-operated Ca2+ entry in HEK-293 cells and its impact on the interpretation of data from stable clones expressing exogenous Ca2+ channels. Cell Calcium, 27, 61-73.

Wu X, Babnigg G and Villereal ML. (2000). Functional significance of human trp1 and trp3 in store-operated Ca2+ entry in HEK-293 cells. Am. J. Physiol. Cell Physiol., 278, C526-C536.

Wu X, Babnigg G, Zagranichnaya T and Villereal ML. (2002). The role of endogenous human trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells. J. Biol. Chem., 277, 13597-13608.

Babnigg G, Zagranichnaya T, Wu X and Villereal ML. (2003). Differential tyrosine phosphorylation of PMCA and regulation of calcium pump activity by carbachol and bradykinin. J. Biol. Chem., 278: 14872-82.

Shalabi A, Zamudio F, Wu X, Scaloni A, Possani LD, Villereal ML. (2004). Tetrapandins, a new class of scorpion toxins that specifically inhibit store-operated calcium entry in human embryonic kidney-293 cells. J. Biol. Chem. 279: 1040-9.