Sonic Hedgehog Stimulates Mouse Embryonic Stem Cell Proliferation By Cooperation Of Ca²⁺/Protein Kinase C and EGF receptor As Well As Gli1 Activation

¹ Department of Veterinary Physiology, Biotherapy Human Resources Center (BK 21), College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea.

^* To whom correspondence should be addressed. E-mail: hjhan{at}chonnam.ac.kr.

	Abstract

Hedgehog signaling has an essential role in the control of stem cell growth in embryonic tissues. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self-renewal of mouse embryonic stem (ES) cells and its related mechanisms. Shh increased DNA synthesis blocked by the inhibition of the smoothened (Smo) receptor. Shh required Gli1 activation to induce the increases in Notch/Hes-1 and Wnt/-catenin. Shh increased the intracellular calcium concentration ([Ca²⁺]_i) and protein kinase C (PKC) activity. We show that the Shh-induced increase in the Gli1 mRNA level requires [Ca²⁺]_i and PKC. Shh increased the phosphorylation of EGF receptor (EGFR), which is blocked by the matrix metalloproteinase (MMP) inhibitor. Subsequently, Shh increased the NF-B p65 phosphorylation, which was inhibited by blocking PKC and EGFR tyrosine kinase. Shh also increased the level of the cell cycle regulatory proteins in a dose-dependent manner. However, Shh decreased the levels of the CDK (cyclin-dependent kinase) inhibitory proteins. The effect of Shh on these proteins was inhibited by blocking PKC, EGFR, and NF-B as well as transfection of Gli1 siRNA, Finally, Shh-induced the progression of the G1/S phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, Shh stimulates mouse ES cell proliferation through Gli1 activation as well as Ca²⁺/PKC and EGFR.

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