Regulation of mesangial cell alpha-smooth muscle actin expression in 3-dimensional matrix by high glucose and growth factors

Periodical: Nephron Exp Nephrol ISBN: 1660-2129 (Electronic)  Number: 2  Date: 2008/07/05  Language: eng  Pages: e46-56

Authors:Whiteside, C., Munk, S., Ispanovic, E., Wang, H., Goldberg, H., Kapus, A., Xia, L.

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Abstract
BACKGROUND/AIMS: We postulated that alpha-smooth muscle actin expressed in primary cultured mesangial cells is down-regulated in 3-dimensional (D) culture and up-regulated by high glucose and growth factors. METHODS: Primary rat mesangial cells were growth-arrested in 5.6 mM (NG) or 30 mM (HG) glucose for 14 days in 3-D Matrigel. Alpha-SM actin expression was analyzed by immunoblotting, real-time PCR and by alpha-SM actin promoter activity in response to 24 h stimulation with endothelin-1 (ET-1), angiotensin II (Ang II) or HG. RESULTS: Alpha-SM actin mRNA, protein and promoter activity were reduced to significantly lower levels in 3-D cells compared to cells in 2-D. Up-regulation of alpha-SM expression was stimulated by ET-1, Ang II and HG. Specific inhibitors of protein kinase C (PKC)-alpha, -beta or -zeta prevented alpha-SM upregulation in HG. In NG, PKC and ERK1/2 activation were required for alpha-SM actin accumulation in 3-D in response to ET-1 or Ang II. In HG, enhanced expression of alpha-SM actin in response to ET-1 or Ang II was unchanged during PKC or ERK1/2 inhibition. CONCLUSION: Mesangial cells in 3-D express low levels of alpha-SM actin representing a more differentiated state. Regulation of alpha-SM actin expression is dependent on specific PKC isozyme and ERK1/2 signaling.