Calcineurin Mediates the Calcium-dependent Inhibition of Adipocyte Differentiation in 3T3-L1 Cells

Abstract

Recent studies have revealed that the calciumdependent serine/threonine phosphatase calcineurin mediates the effects of intracellular calcium in many different cell types. In this study we investigated the role of calcineurin in the regulation of adipocyte differentiation. We found that the specific calcineurin inhibitors cyclosporin A and FK506 overcame the antiadipogenic effect of calcium ionophore on the differentiation of 3T3-L1 preadipocytes. This finding suggests that calcineurin is responsible for mediating the previously documented Ca 2؉ -dependent inhibition of adipogenesis. We further demonstrate that the expression of a constitutively active calcineurin mutant potently inhibits the ability of 3T3-L1 cells to undergo adipocyte differentiation by preventing expression of the proadipogenic transcription factors peroxisome proliferator-activated receptor ␥ (PPAR␥) and CCAAT/enhancer-binding protein ␣ (C/EBP␣). This calcineurin-mediated block in adipocyte differentiation is rescued by ectopic expression of PPAR␥1. Finally, we demonstrate that inhibition of endogenous calcineurin activity with either FK506 or a specific calcineurin inhibitory peptide enhances differentiation of 3T3-L1 cells in response to suboptimal adipogenic stimuli, suggesting that endogenous calcineurin activity normally sets a signaling threshold that antagonizes efficient adipocyte differentiation. Collectively, these data indicate that calcineurin acts as a Ca 2؉ -dependent molecular switch that negatively regulates commitment to adipocyte differentiation by preventing the expression of critical proadipogenic transcription factors.

Prostaglandin F2α inhibits adipocyte differentiation via a Gαq‐Calcium‐Calcineurin‐Dependent signaling pathway

“…Our principal finding is that PGF2a inhibits adipocyte differentiation via a Gaq Gprotein subunit and calcium-dependent signaling pathway involving the calcium/calmodulinregulated serine/threonine phosphatase calcineurin. This conclusion is consistent with previous reports that PGF2a can act via its specific Gq-coupled FP prostanoid receptor to activate calcineurin in a number of cell types [Horsley and Pavlath, 2003;Liang et al, 2004], as well as with our recent identification of calcineurin as a critical calcium-dependent negative regulator of adipocyte differentiation [Neal and Clipstone, 2002]. We presume that this pathway is likely to operate in vivo to influence adipocyte development under conditions where PGF2a levels are elevated, such as those that occur during chronic inflammatory conditions as a result of increased expression of cyclooxygenase-2.…”

“…The pMSCV-GFP, pMSCV-H2K, pMSCVPPARg, pMSCV-VIVIT-GFP retroviral expression vectors were created as previously described [Neal and Clipstone, 2002]. pMSCV-GFP-RGS2 was generated by introducing the GFP-RGS2 fusion sequence from pEGFP-C1-mRGS2 [Reif and Cyster, 2000] into pMSCV-H2K.…”

“…Since treatment of 3T3-L1 preadipocytes with PGF2a has previously been shown to elicit an increase in [Ca 2þ ] i , and elevated [Ca 2þ ] i is known to inhibit adipocyte differentiation [Ntambi and Takova, 1996;Neal and Clipstone, 2002], we next evaluated whether the antiadipogenic effects of PGF2a were mediated by changes in [Ca 2þ ] i and the subsequent activation of downstream Ca 2þ -dependent signaling events. Thus, 3T3-L1 preadipocytes were induced to undergo MDI-induced differentiation in the presence of PGF2a, together with either SKF96365, an inhibitor of receptormediated plasma membrane Ca 2þ -influx, or BAPTA/AM, a membrane-permeable Ca 2þ -chelator.…”

“…Since it appears that the PGF2a-mediated inhibition of adipocyte differentiation is, at least in part, dependent upon changes in the [Ca 2þ ] i , and we have recently identified the calciumregulated serine/threonine phosphatase, calcineurin as a critical component of a Ca 2þ -dependent signaling pathway involved in the negative regulation of adipocyte differentiation [Neal and Clipstone, 2002], we next evaluated whether calcineurin might play a role in the inhibitory effects of PGF2a on adipocyte differentiation. As shown in Figure 3A, we found that treatment of cells with either FK506 or cyclosporin A, two structurally unrelated, highly specific calcineurin inhibitors, both effectively attenuated the inhibitory effects of PGF2a on adipocyte differentiation, as determined by Oil Red O staining.…”

Hypoadiponectinemia Is Associated with Insulin Resistance and Glucose Intolerance after Renal Transplantation

“…This may be explained partly by immunosuppressive therapy (10), in particular steroids (11). Inhibition of endogenous calcineurin activity with tacrolimus or cyclosporine A (CsA) may lead to enhanced adipogenesis, which may explain the metabolic disturbances that are associated with these drugs (12). Some studies have reported linear relationships between doses or blood trough concentrations of CsA and lipids (inverse relationship with HDL) (13,14), but the potential impact of calcineurin inhibitors on serum adiponectin is unknown.…”