KD025 (SLx-2119) suppresses adipogenesis at intermediate stage in human adipose-derived stem cells

Abstract

Rho-associated kinases (ROCKs) have been reported to antagonize adipocyte differentiation, and inhibition of ROCKs by small molecules promotes adipogenesis. Surprisingly, our recent study revealed that the ROCK2-specific inhibitor KD025 (SLx-2119), suppresses differentiation at the intermediate stage in 3T3-L1 preadipocytes. To address whether the anti-adipogenic activity of KD025 is a generalizable property, we examined the effect of KD025 in human adipose-derived stem cells (hADSCs). KD025 significantly suppressed the adipocyte differentiation of hADSCs with downregulation of the protein and mRNA expression of various adipogenic and lipogenic markers, including PPARγ, C/EBPα, SREBP-1c, Glut4 and FABP4. Notably, we observed that adipocyte differentiation is effectively suppressed by exposure to KD025 during the mid-to-late period of adipogenesis but not at the earlier stages, showing stage-specificity. Contrary to expectations, KD025 upregulated the insulin signaling, as confirmed by the increased phosphorylation levels of Akt and GSK-3α/β, and the differentiation-promoting activity of insulin signaling was observed to be overwhelmed by the inhibitory activity. In addition, we observed that other ROCK inhibitors (Y-27632, fasudil, and H-1152P) did not suppress but promoted adipocyte differentiation. These results indicate that KD025 suppresses adipocyte differentiation by modulation of key factors activated at the intermediate stage of differentiation, and not by inhibition of ROCK2.

Keywords: KD025; ROCK; Rho-associated kinase; SLx-2119; adipogenesis; differentiation.

Figure 1.

Measurement of the effect of KD025 on adipogenesis in hADSCs. hADSCs were differentiated by culturing in differentiation media (DM) with or without KD025 at the indicated concentrations. (a) Experimental scheme of differentiation. (b) Cells were stained with ORO at day 15, and microscopic images were taken after the start of differentiation (indicated as day 0). Cells were exposed to 0.3, 0.5, 1, and 3 μM of KD025. (c) Lipid accumulation of (b) was assessed by measuring absorbance at 520 nm. (d) Cells were differentiated with or without 3 μM KD025 until day 24. Microscopic pictures of cells are presented. (e) Lipid accumulation of (d) was assessed by measuring absorbance at 520 nm. **p < 0.01; ***p < 0.001 vs. control.

Figure 2.

Effect of KD025 on fate determination of human stem cells. hADSCs were differentiated with or without 3 μM KD025 for the indicated periods. (a) The number of ORO-stained cells, which are considered adipocytes, were counted in each group from 30 different areas of the obtained microscopic images. (b) All cells were trypsinized, and total number of cells was counted using a hemocytometer. The total number of cells at each time point was expressed as a ratio to the total cell number at the differentiation starting point. *p < 0.05, **p < 0.01; ***p < 0.001 vs. control.

Figure 3.

Effect of KD025 on adipogenic and lipogenic markers. hADSCs were differentiated by incubating in DM with or without 3 μM KD025 over 15-day period. (a) The protein expression levels of PPARγ and FABP4 were analyzed by Western blot, at the indicated time points. β-tubulin was used as a loading control. (b) The expression levels of PPARγ and FABP4 was quantified using the ImageJ software. The relative level was assessed by fold changes compared to day 6/KD025-untreated control cells. (c) The mRNA expression levels of adipogenic genes (PPARG, CEBPA, CEBPB) and lipogenic genes (SLC2A4, SREBF1) at the indicated time points. The relative level was assessed by fold changes compared to KD025-untreated control cells at day 0. *p < 0.05, **p < 0.01; ***p < 0.001 vs. corresponding control condition.

Figure 4.

Stage-specific effect of KD025 on adipogenesis of hADSC. (a) During differentiation of hADSCs, cells were exposed to 3 μM KD025 at various time schedules as indicated, and stained with ORO. The time schedules of inhibition are denoted as the corresponding rods, and the amount of lipid measured was indicated by the relative darkness of the color compared to DM treated group as 100%.(b) Lipid accumulation was assessed by measuring absorbance at 520 nm. The positive control treated for the entire period (from day 0 to day 21) is marked in orange. *p < 0.05, **p < 0.01 vs. positive control (orange box).

Figure 5.

Effects of ROCK inhibitors on insulin signaling. hADSCs were starved in serum-free medium and then stimulated with 20 nM insulin for 20 min. ROCK inhibitors were treated during starvation and stimulation periods. Western blot was performed to evaluate the level of p-Akt (Ser473), p-Akt (Thr308), Akt, and p-GSK-3α/β. β-tubulin was used as the loading control. (b) Band intensity of p-Akt (Ser473) and p-Akt (Thr308) was quantified using the Image J software. The relative level was assessed as fold changes compared to the unstimulated, vehicle-treated control cells. *p < 0.05, **p < 0.01 vs. control.

Figure 6.

Effects of ROCK inhibitors on adipogenesis of hADSCs. (a) During differentiation, cells were exposed to KD025 (3 µM), Y-27632 (5 µM), fasudil (5 µM) and H-1152P (3 µM). Cells were then stained with ORO on day 15. (b) Lipid accumulation was assessed by measuring absorbance at 520 nm. *p < 0.05, **p < 0.01; ***p < 0.001 vs. untreated.

Figure 7.

Proposed model of the mechanistic action of KD025 on the differentiation of hADSCs. KD025 inhibits adipocyte differentiation in hADSCs by suppressing a certain pro-adipogenic regulator. This inactivation overwhelms the pro-adipogenic activity resulting from the Akt activation and ROCK inhibition. Pan-inhibitors promote adipogenesis by activating Akt and suppressing ROCKs.