Hai Yan Huang

BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis

Expression of bone morphogenetic protein 4 (BMP4) in adipocytes of white adipose tissue (WAT) produces “white adipocytes” with characteristics of brown fat and leads to a reduction of adiposity and its metabolic complications. Although BMP4 is known to induce commitment of pluripotent stem cells to the adipocyte lineage by producing cells that possess the characteristics of preadipocytes, its effects on the mature white adipocyte phenotype and function were unknown. Forced expression of a BMP4 transgene in white adipocytes of mice gives rise to reduced WAT mass and white adipocyte size along with an increased number of a white adipocyte cell types with brown adipocyte characteristics comparable to those of beige or brite adipocytes. These changes correlate closely with increased energy expenditure, improved insulin sensitivity, and protection against diet-induced obesity and diabetes. Conversely, BMP4-deficient mice exhibit enlarged white adipocyte morphology and impaired insulin sensitivity. We identify peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) as the target of BMP signaling required for these brown fat-like changes in WAT. This effect of BMP4 on WAT appears to extend to human adipose tissue, because the level of expression of BMP4 in WAT correlates inversely with body mass index. These findings provide a genetic and metabolic basis for BMP4’s role in altering insulin sensitivity by affecting WAT development.

Characterization of adipocyte differentiation from human mesenchymal stem cells in bone marrow

BackgroundAdipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. The mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models (i.e. 3T3-L1 and 3T3-F442A), revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors (C/EBPβ, C/EBPα and PPARγ). However, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells (hBMSCs), remain poorly understood. This study investigated cell cycle regulation and the roles of C/EBPβ, C/EBPα and PPARγ during adipocyte differentiation from hBMSCs.ResultsUtilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPβ, C/EBPα and PPARγ were required for adipocyte differentiation from hBMSCs. C/EBPβ and C/EBPα individually induced adipocyte differentiation in the presence of inducers; PPARγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse.ConclusionsThe characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.

Involvement of cytoskeleton-associated proteins in the commitment of C3H10T1/2 pluripotent stem cells to adipocyte lineage induced by BMP2/4

The developmental pathway that gives rise to mature adipocytes involves two distinct stages: commitment and terminal differentiation. Although the important proteins/factors contributing to terminal adipocyte differentiation have been well defined, the proteins/factors in the commitment of mesenchymal stem cells to the adipocyte lineage cells have not. In this study, we applied proteomics analysis profiling to characterize differences between uncommitted C3H10T1/2 pluripotent stem cells and those that have been committed to the adipocyte lineage by BMP4 or BMP2 with the goal to identify such proteins/factors and to understand the molecular mechanisms that govern the earliest stages of adipocyte lineage commitment. Eight proteins were found to be up-regulated by BMP2, and 27 proteins were up-regulated by BMP4, whereas five unique proteins were up-regulated at least 10-fold by both BMP2/4, including three cytoskeleton-associated proteins (i.e. lysyl oxidase (LOX), translationally controlled tumor protein 1 (TPT1), and αB-crystallin). Western blotting further confirmed the induction of the expression of these cytoskeleton-associated proteins in the committed C3H10T1/2 induced by BMP2/4. Importantly, knockdown of LOX expression totally prevented the commitment, whereas knockdown of TPT1 and αB-crystallin expression partially inhibited the commitment. Several published reports suggest that cell shape can influence the differentiation of partially committed precursors of adipocytes, osteoblasts, and chondrocytes. We observed a dramatic change of cell shape during the commitment process, and we showed that knockdown of these cytoskeleton-associated proteins prevented the cell shape change and restored F-actin organization into stress fibers and inhibited the commitment to the adipocyte lineage. Our studies indicate that these differentially expressed cytoskeleton-associate proteins might determine the fate of mesenchymal stem cells to commit to the adipocyte lineage through cell shape regulation.

Histone demethylase Kdm4b functions as a co-factor of C/EBPβ to promote mitotic clonal expansion during differentiation of 3T3-L1 preadipocytes.

CCAAT/enhancer-binding protein (C/EBP) β is required for both mitotic clonal expansion (MCE) and terminal adipocyte differentiation of 3T3-L1 preadipocytes. Although the role of C/EBPβ in terminal adipocyte differentiation is well defined, its mechanism of action during MCE is not. In this report, histone demethylase Kdm4b, as well as cell cycle genes Cdc45l (cell division cycle 45 homolog), Mcm3 (mini-chromosome maintenance complex component 3), Gins1 (GINS complex subunit 1) and Cdc25c (cell division cycle 25 homolog c), were identified as potential C/EBPβ target genes during MCE by utilizing promoter-wide chromatin immunoprecipitation (ChIP)-on-chip analysis combined with gene expression microarrays. The expression of Kdm4b is induced during MCE and its induction is dependent on C/EBPβ. ChIP, Electrophoretic Mobility Shift Assay (EMSA) and luciferase assay confirmed that the promoter of Kdm4b is bound and activated by C/EBPβ. Knockdown of Kdm4b impaired MCE. Furthermore, Kdm4b interacted with C/EBPβ and was recruited to the promoters of C/EBPβ-regulated cell cycle genes, including Cdc45l, Mcm3, Gins1, and Cdc25c, demethylated H3K9me3 and activated their transcription. These findings suggest a novel feed forward mechanism involving a DNA binding transcription factor (C/EBPβ) and a chromatin regulator (Kdm4b) in the regulation of MCE by controlling cell cycle gene expression.

Transactivation of Atg4b by C/EBPβ Promotes Autophagy To Facilitate Adipogenesis

ABSTRACT Autophagy is a highly conserved self-digestion pathway involved in various physiological and pathophysiological processes. Recent studies have implicated a pivotal role of autophagy in adipocyte differentiation, but the molecular mechanism for its role and how it is regulated during this process are not clear. Here, we show that CCAAT /enhancer-binding protein β (C/EBPβ), an important adipogenic factor, is required for the activation of autophagy during 3T3-L1 adipocyte differentiation. An autophagy-related gene, Atg4b, is identified as a de novo target gene of C/EBPβ and is shown to play an important role in 3T3-L1 adipocyte differentiation. Furthermore, autophagy is required for the degradation of Klf2 and Klf3, two negative regulators of adipocyte differentiation, which is mediated by the adaptor protein p62/SQSTM1. Importantly, the regulation of autophagy by C/EBPβ and the role of autophagy in Klf2/3 degradation and in adipogenesis are further confirmed in mouse models. Our data describe a novel function of C/EBPβ in regulating autophagy and reveal the mechanism of autophagy during adipocyte differentiation. These new insights into the molecular mechanism of adipose tissue development provide a functional pathway with therapeutic potential against obesity and its related metabolic disorders.

Down-regulation of type I Runx2 mediated by dexamethasone is required for 3T3-L1 adipogenesis

Runx2, a runt-related transcriptional factor family member, is involved in the regulation of osteoblast differentiation. Interestingly, it is abundant in growth-arrested 3T3-L1 preadipocytes and was dramatically down-regulated during adipocyte differentiation. Knockdown of Runx2 expression promoted 3T3-L1 adipocyte differentiation, whereas overexpression inhibited adipocyte differentiation and promoted the trans-differentiation of 3T3-L1 preadipocytes to bone cells. Runx2 was down-regulated specifically by dexamethasone (DEX). Only type I Runx2 was expressed in 3T3-L1 preadipocytes. Using luciferase assay and chromatin immunoprecipitation-quantitative PCR analysis, it was found that DEX repressed this type of Runx2 at the transcriptional level through direct binding of the glucocorticoid receptor (GR) to a GR-binding element in the Runx2 P2 promoter. Further studies indicated that GR recruited histone deacetylase 1 to the Runx2 P2 promoter which then mediated the deacetylation of histone H4 and down-regulated Runx2 expression. Runx2 might play its repressive role through the induction of p27 expression, which blocked 3T3-L1 adipocyte differentiation by inhibiting mitotic clonal expansion. Taken together, we identified Runx2 as a new downstream target of DEX and explored a new pathway between DEX, Runx2, and p27 which contributed to the mechanism of the 3T3-L1 adipocyte differentiation.

Transcriptional activation of histone H4 by C/EBPβ during the mitotic clonal expansion of 3T3-L1 adipocyte differentiation

Histone H4 is activated by C/EBPβ in mitotic clonal expansion during adipogenesis. C/EBP-binding sites are identified in histone H4 promoters, and H4 expression is suppressed when C/EBPβ is knocked down or its DNA-binding activity is inhibited by A-C/EBP. These results help in our understanding of how C/EBPβ plays important roles in the proliferation of other cells.

Protein inhibitor of activated STAT 1 (PIAS1) is identified as the SUMO E3 ligase of CCAAT/enhancer-binding protein β (C/EBPβ) during adipogenesis.

ABSTRACT It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein β (C/EBPβ), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPβ has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPβ to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPβ began to decline. PIAS1 was found to interact with C/EBPβ through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPβ. C/EBPβ became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPβ. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPβ.

MicroRNA-140 Promotes Adipocyte Lineage Commitment of C3H10T1/2 Pluripotent Stem Cells via Targeting Osteopetrosis-associated Transmembrane Protein 1

Background: BMP4 treatment induces adipocyte lineage commitment of C3H10T1/2 pluripotent stem cells. Results: Expression of miR-140 increases significantly during adipocyte lineage commitment. Conclusion: miR-140 promotes adipocyte lineage commitment through down-regulating Ostm1. Significance: Which miRNA and how it functions in adipocyte lineage commitment are clarified. BMP4 has been shown to induce C3H10T1/2 pluripotent stem cells to commit to adipocyte lineage. In addition to several proteins identified, microRNAs also play a critical role in the process. In this study, we identified microRNA-140 (miR-140) as a direct downstream component of the BMP4 signaling pathway during the commitment of C3H10T1/2 cells to adipocyte lineage. Overexpression of miR-140 in C3H10T1/2 cells promoted commitment, whereas knockdown of its expression led to impairment. Additional studies indicated that Ostm1 is a bona fide target of miR-140, which is significantly decreased during commitment, and Ostm1 was also demonstrated to function as an anti-adipogenic factor.

Regulation of cell cycle of hepatocellular carcinoma by NF90 through modulation of cyclin E1 mRNA stability

Activation of cyclin E1, a key regulator of the G1/S cell-cycle transition, has been implicated in many cancers including hepatocellular carcinoma (HCC). Although much is known about the regulation of cyclin E1 expression and stability, its post-transcriptional regulation mechanism remains incompletely understood. Here, we report that nuclear factor 90 (NF90), a double-stranded RNA (dsRNA) binding protein, regulates cyclin E1 in HCC. We demonstrate that NF90 is upregulated in HCC specimens and that suppression of NF90 decreases HCC cell growth and delays G1/S transition. We identified cyclin E1 as a new target of NF90 and found a significant correlation between NF90 and cyclin E1 expression in HCC. The mRNA and protein levels of cyclin E1 were downregulated upon NF90 knockdown. Suppression of NF90 caused a decrease in the half-life of cyclin E1 mRNA, which was rescued by ectopic expression of NF90. Furthermore, NF90 bound to the 3’ untranslated regions (3’UTRs) of cyclin E1 mRNA in vitro and in vivo. Knockdown of NF90 also inhibited tumor growth of HCC cell lines in mouse xenograft model. Moreover, we showed that inhibition of NF90 sensitized HCC cells to the cyclin-dependent kinase 2 (CDK2) inhibitor, roscovitine. Taken together, downregulation of NF90 in HCC cell lines can delay cell-cycle progression, inhibit cell proliferation, and reduce tumorigenic capacity in vivo. These results suggest that NF90 has an important role in HCC pathogenesis and that it can serve as a novel therapeutic target for HCC.