BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage - PubMed (original) (raw)

BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage

Haiyan Huang et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. After treatment of C3H10T1/2 stem cells with these BMPs during proliferation followed by exposure to differentiation inducers at growth arrest, nearly all cells enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype. Overexpression of constitutively active BMP receptor (CA)-BMPr1A or CA-BMPr1B induces commitment in the absence of BMP2/4, whereas overexpression of a dominant-negative receptor dominant-negative-BMPr1A suppresses commitment induced by BMP. Also, knockdown of the expression of Smad4 (coregulator in the BMP/Smad signaling pathway) with RNAi disrupts commitment by the BMPs. However, knockdown of expression of p38 MAPK (an intermediary in the BMP/MAPK signaling pathway) with RNAi had little effect on BMP-induced commitment. Together, these findings indicate that the BMP/Smad signaling pathway has a dominant role in adipocyte lineage determination. Proteomic analysis identified lysyl oxidase (LOX), a bona fide downstream target gene of the BMP signaling pathway. Expression of LOX is induced by BMP2/4 during adipocyte lineage commitment, and knockdown of its expression disrupts the commitment process.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Induction of adipocyte lineage commitment. Dependence on BMP2 and BMP4 concentration. C3H10T1/2 pluripotent stem cells at low density, were cultured without or with BMP2 or BMP4 at 10, 25, and 50 ng/mL until postconfluent, and then subjected to the adipocyte differentiation protocol. (A) On day 8, the accumulation of cytoplasmic triglyceride was assessed by Oil Red O staining. (B) On day 4, whole-cell extracts were subjected to SDS/PAGE and 422/aP2 was detected by immunoblotting.

Fig. 2.

Fig. 2.

C3H10T1/2 stem cells express BMP receptors 1A and 2 and respond to BMP signals. (A) C3H10T1/2 stem cells were cultured in DMEM with 10% calf serum. Total RNA was isolated and subjected to RT-PCR. DNA marker (lane 1); BMPr1A (lane 2); BMPr1B (lane 3); and BMPr2 (lane 4). (B) Two days after plating, the cells were treated with 50 ng of BMP2 per mL and equal levels of cell extract protein were subjected to SDS/PAGE and immunoblotted with the indicated antibodies.

Fig. 3.

Fig. 3.

Overexpression of constitutively active BMPr1 (CA-BMPr1A or CA-BMPr1B) induces adipocyte lineage commitment in the absence of BMP. C3H10T1/2 stem cells were infected with retroviral CA-BMPr1A or CA-BMPr1B. (A) One day after infection, total RNA was isolated and subjected to RT-PCR. (B) Cell lysates were prepared, and equal levels of extract protein were separated by SDS/PAGE and immunoblotted with the antibodies indicated. (C) After reaching postconfluence, the cells were induced to differentiate, and on day 12, cells were stained. (D) On day 4, cell extracts were prepared, and the expression of 422/aP2 was assessed by immunoblotting.

Fig. 4.

Fig. 4.

Overexpression of DN-BMPr1A prevents adipocyte lineage commitment induced by BMP. C3H10T1/2 stem cells were infected with retrovirus harboring DN-BMPr1A. (A) One day after infection, total RNA was isolated and subjected to RT-PCR to confirm the expression of DN-BMPr1A; β-actin was used as loading control. (B) Whole-cell lysates were prepared, and equal amounts of protein were separated by SDS/PAGE and immunoblotted with antibodies as indicated. (C) Upon reaching postconfluence, cells were induced to differentiate with standard adipocyte differentiation protocol, and on day 8, were stained with Oil Red O. (D) On day 4, cell extracts were prepared and the expression of 422/aP2 was assessed by immunoblotting.

Fig. 5.

Fig. 5.

Dependence of stem cell commitment on Smad signaling. C3H10T1/2 stem cells were plated at 30% confluence and transfected with Smad4 Stealth RNAi, and 24 h later were treated with BMP2 (50 ng/mL) until postconfluent. Cells were then induced to differentiate with the adipocyte differentiation protocol. Knockdown of Smad4 expression was confirmed by immunoblotting (B), and its effect on adipocyte commitment and terminal differentiation was assessed both by staining of cytoplasmic triglyceride (A) and by expression of 422/aP2 (C).

Fig. 6.

Fig. 6.

Dependence of stem cell commitment on p38MAPK signaling. (A and B) C3H10T1/2 stem cells were plated at low density, treated with a specific p38MAPK inhibitor SB203580 (10 μM) before treatment with BMP2 (50 ng/mL). After reaching postconfluence, cells were induced to differentiate. Commitment and terminal differentiation were assessed by: staining of cytoplasmic triglyceride (A) and expression of an adipocyte marker protein (422/aP2) (B). (C–E) C3H10T1/2 stem cells were plated at 30% confluence and transfected with p38MAPK Stealth RNAi. After 24 h, cells were treated with BMP2 (50 ng/mL) until reaching postconfluence, and then were induced to differentiate using the adipocyte differentiation protocol. (C) Knockdown of expression of p38MAPK was verified by immunoblotting. (D and E) The effect on commitment and terminal differentiation was assessed both by staining of cytoplasmic triglyceride (D) and by expression of specific adipocyte marker protein (422/aP2) (E).

Fig. 7.

Fig. 7.

Role of LOX in the BMP-induced commitment to the adipocyte lineage. (A) Effect of BMP2 and BMP4 on the induction of LOX expression. C3H10T1/2 stem cells were plated at 30% confluence and after 24 h were treated with 50 ng/mL of BMP2 or BMP4 until postconfluence. (B) Expression of LOX was assessed by immunoblotting. C3H10T1/2 cells were plated as above and transfected with LOX Stealth RNAi. (C and D) After 24 h, cells were treated with BMP2 (50 ng/mL) until postconfluent, at which time the expression of LOX was measured and the effect on adipocyte commitment and terminal differentiation was assessed both by staining of cytoplasmic triglyceride (C) and expression of an adipocyte-specific marker (422/aP2) (D). (E) Evidence that LOX is a downstream target gene of BMP signaling pathway. C3H10T1/2 stem cells were plated at 30% confluence, transfected with Smad4 or p38MAPK RNAi 24 h later, cultured without or with BMP until postconfluent, cell extracts were prepared, and the effect on Smad4, p38MAPK and LOX expression was assessed by immunoblotting.

Scheme 1.

Scheme 1.

Adipocyte lineage commitment and terminal differentiation.

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