Functional Transient Receptor Potential Channels in Human Preadipocytes and Cardiac C-Kit⁺ Progenitor Cells

This dissertation, "Functional Transient Receptor Potential Channels in Human Preadipocytes and Cardiac C-kit⁺ Progenitor Cells" by Hui, Che, 車慧, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Transient receptor potential (TRP) channels play important roles in cellular physiology and biology. The present PhD project investigated the functional expression of TRPV and TRPM channels in human preadipocytes and cardiac c-kit+ progenitor cells and their roles in regulating cell proliferation, adipogenic differentiation or migration. In addition, the role of store-operated Ca2+ entry (SOCE) channels in regulating cell proliferation and migration was also studied in human cardiac c-kit+ progenitor cells using multiple approaches including whole-cell patch voltage-clamp, confocal microscope, molecular biology, etc. We found that TRPV2, TRPV4 and TRPM7 channels were abundantly expressed in human preadipocytes. Activation of TRPV2 channels by probenecid caused a long-lasting intracellular Ca2+ transient, while activation of TRPV4 channels by 4-PDD induced Ca2+ oscillations. TRPM7 current was recorded with a Mg2+-free pipette solution, and inhibited by 2-aminoethyl diphenyl borate (2-APB). Silence of TRPV2 or TRPM7, but not TRPV4, with the specific shRNA, reduced cell proliferation via inhibiting cyclin D1, cyclin E, and p-ERK1/2. Individually silencing these three channels decreased adipogenic differentiation by reducing p-Akt kinase. The results indicate that TRPV2, TRPV4 and TRPM7 are involved in adipogenesis, while TRPV2 and TRPM7, but not TRPV4, regulate cell proliferation in human preadipocytes. In second part of the thesis, abundant expression of TRPV2, TRPV4, and TRPM7 channels was demonstrated in human cardiac c-kit+ progenitor cells. Similar to human preadipocytes, probenecid and 4-PDD activated Ca2+ signaling, and TRPM7 current recorded with a Mg2+-free pipette solution was inhibited by 2-APB. Silencing TRPV2 or TRPM7, but not TRPV4, inhibited cell proliferation by arresting cells at G0/G1 phase with a reduced cyclin D, cyclin E, and p-ERK1/2. Cell migration was decreased with silence of TRPV2, TRV4 or TRPM7 via inhibiting p-Akt kinase. The results show that TRPV2, TRPV4 and TRPM7 mediate cell migration, while TRPV2 and TRPM7, but not TRPV4 channels, participate in regulating cell proliferation. In third part of the thesis, we demonstrated that SOCE channels were composed of TRPC1, STIM1 and Orai1 by protein-protein interaction. Silence of TRPC1, STIM1, or Orai1 with specific siRNA reduced Ca2+ influx through SOCE channels, decreased cell proliferation by inhibiting cyclin D1 and cyclin E, and slowed down cell migration via reducing p-Akt kinase. These results suggest that TRPC1, STIM1 and Orai1 are the major components of SOCE channels in human cardiac c-kit+ cells. SOCE channels play an essential role in regulating cell proliferation and migration. Collectively, this PhD project has demonstrated for the first time that 1) TRPV2, TRPV4, and TRPM7 are abundantly expressed in human preadipocytes and cardiac c-kit+ progenitor cells. 2) These TRP channels regulate adipogenic differentiation in preadipocytes and migration in cardiac c-kit+ progenitor cells. 3) TRPV2 and TRPM7, but not TRPV4, are involved in cell proliferation of human preadipocytes and cardiac c-kit+ progenitor cells. 4) TRPC1, STIM1 and Orai1 are interacted to form SOCE channels and regulate cell proliferation and migration in human cardiac c-kit+ cells. 5) All the above physiological roles of TRPV2, TRPV4, TRPM7, and SOCE channels are mediated by cyclin D1,