MITF and PU.1 inhibit adipogenesis of ovine primary preadipocytes by restraining C/EBPβ

Abstract

Background: PU box-binding protein (PU.1) is a master gene of hematopoietic lineage and an important specific transcription factor in osteoclast lineage. There is proof of its expression in adipose tissue, and it is known to significantly and negatively affect adipogenesis. However, it is unclear whether there are any other molecules involved in this process.

Methods: We wished to explore the effect of PU.1's co-activator microphthalmia-associated transcription factor (MITF) on the adipogenic differentiation of ovine primary preadipocytes. The expression vectors pcDNA-MITF and pcDNA-PU.1, and MITF siRNA and PU.1 siRNA were transfected or co-transfected into ovine tail primary preadipocytes. Real-time PCR and western blot analysis were applied to investigate the expression levels of PU.1 and MITF. The morphologic changes in the cells were observed under a microscope at a magnification of × 200 after staining with Oil Red O. The triglyceride (TG) content in cells was also determined after transfection.

Results: MITF and its co-activator PU.1 synergistically exhibited an opposite expression pattern to that of CCAAT-enhancer-binding protein-β (C/EBPβ) during adipogenic differentiation of ovine primary preadipocytes. Before induction of differentiation, overexpression of MITF or PU.1 inhibited the expression of C/EBPβ and adipogenesis in the cells; and knockdown of MITF or PU.1 promoted the expression of C/EBPβ and adipogenesis in the cells. The inhibitory or promotive effect was enhanced when MITF and PU.1 were co-overexpressed or co-silenced. However, when MITF and/or PU.1 were overexpressed after day 2 of differentiation, no changes in adipogenesis of the cells were observed.

Conclusions: MITF and its co-activator PU.1 inhibited adipogenesis of ovine primary preadipocytes by restraining C/EBPβ.

Keywords: Adipogenesis; CCAAT-enhancer-binding protein-β; Lineage-specific transcription factor; Microphthalmia-associated transcription factor; PU box-binding protein.

Fig. 1

Overexpression of MITF suppressed the expression of C/EBPβ and PPARγ in ovine primary preadipocytes. Primary preadipocytes were isolated from 20-day old small, fat-tailed sheep using the collagenase I digestion method. The cells were seeded in culture plates at a density of 5 × 10⁴ cells/cm² and cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were then induced to differentiate using the “cocktail” method. On days 0, 2, 4, 6 and 8 during adipogenic differentiation, the cells were collected and their total RNA and proteins were extracted. The expression levels of MITF, PU.1, C/EBPβ and PPARγ were detected via qPCR and western blotting analyses. a – The mRNA expression profiles of MITF, PU.1, C/EBPβ and PPARγ during adipogenic differentiation. The mRNA levels of β-tubulin on days 0, 2, 4, 6 and 8 were used as the reference for fold change calculation at each time point. b – The protein expression profiles of MITF, PU.1, C/EBPβ and PPARγ during adipogenic differentiation. c – Protein expression of C/EBPβ and PPARγ was suppressed by pcDNA-MITF transfection. d – Oil Red O staining for the ovine primary preadipocytes transfected with pcDNA-MITF on day 6 of differentiation

Fig. 2

Overexpression of MITF and its co-activator PU.1 inhibited adipogenesis of ovine primary preadipocytes. On reaching 80% confluence, the primary cells were transfected with pcDNA-MITF, pcDNA-PU.1, pcDNA-MITF and pcDNA-PU.1, or nothing. Then the cells were induced to differentiate. On day 6 of adipogenic differentiation, their total proteins were extracted. a – Expression of MITF, PU.1, C/EBPβ, PPARγ and aP2 after pcDNA-MITF and/or pcDNA-PU.1 transfection. b – Oil Red O staining for the ovine primary preadipocytes transfected with pcDNA-MITF and/or pcDNA-PU.1 on day 6 of differentiation. The scale bar is 200 μm. TG content change is shown in (c) (510 nm OD value) and (d) (μmol/ng protein) after primary preadipocytes were transfected with pcDNA-MITF and/or pcDNA-PU.1 on day 6 of differentiation. The same letters on the bar diagram indicated no significant differences between datasets; significant differences indicated by different letters. Significance was set at p < 0.05 (n = 3), *p < 0.05 vs. the control, ^# p < 0.05 vs. pc-MITF

Fig. 3

Knockdown of MITF and PU.1 promoted adipogenesis of ovine primary preadipocytes. On reaching 80% confluence, the primary cells were transfected with MITF siRNA, PU.1 siRNA, MITF siRNA and PU.1 siRNA, or nothing. Then the cells were induced to differentiate. On day 6 of adipogenic differentiation, their total proteins were extracted. a – Expression of MITF, PU.1, C/EBPβ, PPARγ and aP2 after MITF siRNA and/or PU.1 siRNA transfection. b – Oil Red O staining for the ovine primary preadipocytes transfected with MITF siRNA and/or PU.1 siRNA on day 6 of differentiation; The scale bar is 200 μm. The bar diagram (bottom right) shows the OD value (510 nm) of the cell matrix containing ovine primary preadipocytes transfected with MITF siRNA and/or PU.1 siRNA on day 6 of differentiation. TG content change is shown in (c) (510 nm OD value) and (d) (μmol/ng protein) after the primary preadipocytes were transfected with MITF siRNA and/or PU.1 siRNA on day 6 of differentiation. The same letters on the bar diagram indicated no significant differences between datasets; significant differences indicated by different letters. Significance was set at p < 0.05 (n = 3), *p < 0.05 vs. control, ^# p < 0.05 vs. siMITF

Fig. 4

Overexpression of MITF and PU.1 after day 2 of differentiation did not affect the adipogenesis of the ovine primary preadipocytes. The primary preadipocytes were induced to differentiate. On day 2 of differentiation after the induction solution 1 was changed for 4 h, pcDNA-MITF and pcDNA-PU.1 were transfected alone or co-transfected into the cells. Oil Red O staining and TG content analyses were applied to examine the lipid accumulation in the cells on day 6. a – Oil Red O staining for the cells on day 6 of differentiation. The scale bar is 200 μm. The bar diagram (bottom left) showing the OD value (510 nm) of the cell matrix containing ovine primary preadipocytes transfected with MITF siRNA and/or PU.1 siRNA on day 6 of differentiation. b – TG content analysis for cells on day 6 of differentiation (510 nm OD value). c – TG content analysis for cells on day 6 of differentiation (umol/ng protein). The same letters on the bar diagram indicated no significant differences between datasets; significant differences indicated by different letters. Significance was set at p < 0.05 (n = 3), *p < 0.05 vs. control, ^# p < 0.05 vs. siMITF. d. Expression of MITF, PU.1, C/EBPβ and PPARγ after pcDNA-MITF and/or pcDNA-PU.1 transfection