Mechanical loading regulates NFATc1 and beta-catenin signaling through a GSK3beta control node

Abstract

Mechanical stimulation can prevent adipogenic and improve osteogenic lineage allocation of mesenchymal stem cells (MSC), an effect associated with the preservation of beta-catenin levels. We asked whether mechanical up-regulation of beta-catenin was critical to reduction in adipogenesis as well as other mechanical events inducing alternate MSC lineage selection. In MSC cultured under strong adipogenic conditions, mechanical load (3600 cycles/day, 2% strain) inactivated GSK3beta in a Wnt-independent fashion. Small interfering RNA targeting GSK3beta prevented both strain-induced induction of beta-catenin and an increase in COX2, a factor associated with increased osteoprogenitor phenotype. Small interfering RNA knockdown of beta-catenin blocked mechanical reduction of peroxisome proliferator-activated receptor gamma and adiponectin, implicating beta-catenin in strain inhibition of adipogenesis. In contrast, the effect of both mechanical and pharmacologic inhibition of GSK3beta on the putative beta-catenin target, COX2, was unaffected by beta-catenin knockdown. GSK3beta inhibition caused accumulation of nuclear NFATc1; mechanical strain increased nuclear NFATc1, independent of beta-catenin. NFATc1 knockdown prevented mechanical stimulation of COX2, implicating NFATc1 signaling. Finally, inhibition of GSK3beta caused association of RNA polymerase II with the COX2 gene, suggesting transcription initiation. These results demonstrate that mechanical inhibition of GSK3beta induces activation of both beta-catenin and NFATc1 signaling, limiting adipogenesis via the former and promoting osteoblastic differentiation via NFATc1/COX2. Our novel findings suggest that mechanical loading regulates mesenchymal stem cell differentiation through inhibition of GSK3beta, which in turn regulates multiple downstream effectors.

FIGURE 1.

Mechanical effect on β-catenin is critical for inhibition of adipogenesis. A, C3H10T1/2 cells in adipogenic medium were treated with or without DKK-1 (50 ng/ml), and strain was applied daily for 4 days. Total cellular proteins were immunoblotted for adiponectin (APN), PPARγ, β-catenin (β-cat) both total and active, and tubulin as designated. B, DKK-1 (50 ng/ml, added 30 min prior to Wnt3a) prevents Wnt3a (100 ng/ml) stimulation of GSK3β phosphorylation and increase in active β-catenin measured at 3 h. C, DKK-1 does not prevent GSK3β phosphorylation induced by 1 h of mechanical strain. D, β-catenin mRNA and protein shown 48 h after addition of siCat or siScr (CTL). E, 48 h after the addition of siScr or siCat, the strain regimen (left panel) or SB415286 (20 μm, right panel, noted as SB) was applied for 4 days. Adiponectin mRNA was amplified by real time RT-PCR. For mRNA experiments, significant change from control condition is shown by an asterisk, p < 0.01.

FIGURE 2.

Mechanical stimulation of COX2 does not require β-catenin activation. A, after exposure to daily strain regimen for 4 days, mRNA was amplified by real time RT-PCR for COX2, adiponectin and Wisp1. B, proteins were analyzed by immunoblot after treatment as in A. C, proteins are shown after treatment with siRNA ± daily strain. Densitometry for adiponectin bands collated from three separate experiments are shown below the blots where the strain condition (gray bars) is shown compared with the unstrained control (CTL) for each siRNA (black bars); a = different from unstrained control, p < 0.01 by analysis of variance. D, cells were pretreated with siRNA followed by 4 days with or without SB415286 (20 μm). Densitometry of adiponectin bands from three separate experiments is shown below, with presentation as in C. a = different from unstrained control, p < 0.01; b = different from unstrained control and from a, p < 0.05.

FIGURE 3.

GSK3β is involved in regulation of both adiponectin and COX2. A, GSK3β protein was silenced with targeted siRNA prior to daily strain regimen. The immunoblot for designated proteins is shown after 4 days; tubulin serves as a loading control. B, experiment as in A, except treatment was ± SB415286 (20 μm). C, 24 h after application of siRNA targeting β-catenin, or GSK3β, as noted, the daily strain regimen was applied for 2 days, and the lysates were analyzed. COX2 protein is increased by mechanical load when β-catenin is knocked down, but not when GSK3β is silenced. D, similar to C, the stimulation of COX2 protein by SB415286 is prevented when GSK3β is knocked down.

FIGURE 4.

Mechanical strain promotes NFATc1 nuclear accumulation and increases in COX2 by inhibition of GSK3β. A, mechanical strain was applied to cells for the indicated time. Nuclear NFATc1 bands are shown in the top row, with PARP to verify nuclear origin of the sample. COX2, analyzed from cytoplasmic lysates (lactate dehydrogenase (LDH) marker verifying cytoplasmic origin shown below), rises during application of strain. B, cells were treated with SB415286 (20 μm), and nuclear and cytoplasmic fractions were collected to show the rise in nuclear NFATc1 when GSK3β is inhibited. C, siRNA knockdown of GSK3β, shown in the top row, prevents strain effect on NFATc1 and COX2, measured 4 h after initiating the strain regimen. Mechanical strain increased both nuclear NFATc1 and cytoplasmic COX2 at 4 h. D, siRNA knockdown of β-catenin does not prevent strain induced nuclear accumulation of NFATc1, or the increase in COX2. E, as in D, both NFATc1 and COX2 stimulation with SB415286 are unaffected by β-catenin knockdown.

FIGURE 5.

Inhibition of calcium/calcineurin signaling does not block strain induced NFATc1 nuclear accumulation. A, cells were treated with ionomycin (1 μm) the for indicated times, and proteins from nuclear and cytoplasmic fractions were immunoblotted for proteins as shown. An effect to stimulate calcineurin is assured by increase in nuclear NFATc1, with consequent increase in COX2. B, cells were pretreated with tacrolimus (5 μm) to prevent ionomycin NFATc1 activation; tacrolimus inhibits both the ionomycin stimulated NFATc1 translocation and the increase in COX2 measured at 4 h. C, pretreatment with tacrolimus does not prevent either nuclear accumulation of NFATc1 nor rise in cytosolic COX2 because of strain at 4 h. D, cells were pretreated with tacrolimus and then SB415286 for 4 h prior to collection of nuclear and cytosolic protein for immunoblot. Tacrolimus did not prevent either NFATc1 nuclear translocation or COX2 increase induced by SB415286.

FIGURE 6.

NFATc1 is critical to mechanical stimulation of COX2, but not for mechanical repression of adipogenesis. A, NFATc1 was targeted with siRNA and cell proteins analyzed 4 h after strain application. In the presence of NFATc1 knockdown, COX2 did not respond to strain. B, similarly, after NFATc1 knockdown, SB415286 (20 μm) failed to cause COX2 increase. C, cells treated with siRNA (NFATc1 or negative control siRNA) were cultured for 4 days in adipogenic medium with daily application of strain. Strain still effectively decreased adipogenesis as shown by decreases in both adiponectin (APN) and PPARγ. D, similar to C, cells exposed to SB415286 during the 4-day exposure to the adipogenic medium had a decrease in adipogenesis unaffected by knockdown of NFATc1.

FIGURE 7.

mdMSC respond to mechanical strain with decreased adipogenesis and increased COX2 dependent on mechanical inhibition of GSK3β. A, microphotograph (40×, gray scale) of mdMSC in adipogenic medium with or without strain show mechanical strain reduces Oil-Red O stain of intracellular lipid consistent with adipocyte phenotype. B, Western blot of cells treated with siRNA either scrambled (−) or targeting β-catenin (+) shows that siRNA knockdown of β-catenin prevents strain inhibition of adiponectin (APN), whereas the mechanical effect to increase COX2 is unperturbed. C, GSK3β knockdown with siRNA disrupts adipogenesis, and β-catenin is not activated by strain. D, GSK3β knockdown prevents strain-induced NFATc1 nuclear accumulation as well as COX2 expression.

FIGURE 8.

GSK3β inhibition increases COX2 gene expression. The COX2 promoter luciferase reporter construct was transfected into C3H10T1/2 cells along with a β-galactosidase reporter. The data are shown as luciferase activity corrected for β-galactosidase transfection. The cells were treated with indicated reagents for 24 h prior to measuring luciferase/β-galactosidase. A, no changes in COX2 promoter activity were seen because of either ionomycin (1 μm) or SB415286 (20 μm). B, COX2 promoter activity (corrected for β-galactosidase) was not stimulated by ionomycin or phorbol 12-myristate 13-acetate (15 ng/ml) but was increased by a combination of ionomycin + phorbol 12-myristate 13-acetate as well as prostaglandin E2 (10 μm). C, cells were treated with or without SB415286 for 24 h and then treated with actinomycin D (1 μm) for indicated time. COX2 mRNA was amplified by real time RT-PCR showed that COX2 t_½ was not changed by GSK3β inhibition. D, MSC were treated ± SB415286 for 24 h and chromatin-immunoprecipitated (IP) with RNA polymerase II antibody. IgG was used as a negative control (CTL). The COX2 gene (−136 to +159) amplified by PCR is associated with polymerase II after GSK3β inhibition.