Abstract
Background/Aims: We postulated that α-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 α-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 α-SM expression was stimulated by ET-1, Ang II and HG. Specific inhibitors of protein kinase C (PKC)-α, -β or -ζ prevented α-SM upregulation in HG. In NG, PKC and ERK1/2 activation were required for α-SM actin accumulation in 3-D in response to ET-1 or Ang II. In HG, enhanced expression of α-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 α-SM actin representing a more differentiated state. Regulation of α-SM actin expression is dependent on specific PKC isozyme and ERK1/2 signaling.
Journal Section:
Original Paper
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