Sona Kang

Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ

Mesenchymal precursor cells have the potential to differentiate into several cell types, including adipocytes and osteoblasts. Activation of Wnt/β-catenin signaling shifts mesenchymal cell fate toward osteoblastogenesis at the expense of adipogenesis; however, molecular mechanisms by which Wnt signaling alters mesenchymal cell fate have not been fully investigated. Our prior work indicates that multipotent precursors express adipogenic and osteoblastogenic transcription factors at physiological levels and that ectopic expression of Wnt10b in bipotential ST2 cells suppresses expression of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and increases expression of Runx2, Dlx5, and osterix. Here, we demonstrate that transient activation of Wnt/β-catenin signaling rapidly suppresses C/EBPα and PPARγ, followed by activation of osteoblastogenic transcription factors. Enforced expression of C/EBPα or PPARγ partially rescues lipid accumulation and decreases mineralization in ST2 cells expressing Wnt10b, suggesting that suppression of C/EBPα and PPARγ is required for Wnt/β-catenin to alter cell fate. Furthermore, knocking down expression of C/EBPα, PPARγ, or both greatly reduces adipogenic potential and causes spontaneous osteoblastogenesis in ST2 cells and mouse embryonic fibroblasts, suggesting that Wnt signaling alters the fate of mesenchymal precursor cells primarily by suppressing C/EBPα and PPARγ.

Wnt10b Inhibits Development of White and Brown Adipose Tissues

Wnt is a family of secreted signaling proteins that regulate diverse developmental processes. Activation of canonical Wnt signaling by Wnt10b inhibits differentiation of preadipocytes in vitro. To determine whether Wnt signaling blocks adipogenesis in vivo, we created transgenic mice in which Wnt10b is expressed from the FABP4 promoter. Expression of Wnt10b in adipose impairs development of this tissue throughout the body, with a decline of ∼50% in total body fat and a reduction of ∼60% in weight of epididymal and perirenal depots. FABP4-Wnt10b mice resist accumulation of adipose tissue when fed a high fat diet. Furthermore, transgenic mice are more glucose-tolerant and insulin-sensitive than wild type mice. Expression of Wnt10b from the FABP4 promoter also blocks development of brown adipose tissue. Interscapular tissue of FABP4-Wnt10b mice has the visual appearance of white adipose tissue but expresses neither brown (e.g. uncoupling protein 1) nor white adipocyte markers. Transgenic mice are unable to maintain a core body temperature when placed in a cold environment, providing further evidence that Wnt10b inhibits development of brown adipose tissue. Although food intake is not altered in FABP4-Wnt10b mice, oxygen consumption is decreased. Thus, FABP4-Wnt10b mice on a chow diet gain more weight than controls, largely because of an increase in weight of skin. In summary, inhibition by Wnt10b of white and brown adipose tissue development results in lean mice without lipodystrophic diabetes.

Role of wnts in prostate cancer bone metastases

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor‐induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine‐rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP‐produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf‐1 (DKK‐1). It appears that DKK‐1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK‐1 expression decreases allowing for unmasking of Wnts osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK‐1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK‐1 activity fits a model of CaP‐induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone‐related protein (PTHRP) and DKK‐1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK‐1); and an osteoblastic phase, in which tumor growth‐associated hypoxia results in production of vascular endothelial growth factor and endothelin‐1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases. J. Cell. Biochem. 97: 661–672, 2006.

IRF4 is a key thermogenic transcriptional partner of PGC-1α.

Brown fat can reduce obesity through the dissipation of calories as heat. Control of thermogenic gene expression occurs via the induction of various coactivators, most notably PGC-1α. In contrast, the transcription factor partner(s) of these cofactors are poorly described. Here, we identify interferon regulatory factor 4 (IRF4) as a dominant transcriptional effector of thermogenesis. IRF4 is induced by cold and cAMP in adipocytes and is sufficient to promote increased thermogenic gene expression, energy expenditure, and cold tolerance. Conversely, knockout of IRF4 in UCP1(+) cells causes reduced thermogenic gene expression and energy expenditure, obesity, and cold intolerance. IRF4 also induces the expression of PGC-1α and PRDM16 and interacts with PGC-1α, driving Ucp1 expression. Finally, cold, β-agonists, or forced expression of PGC-1α are unable to cause thermogenic gene expression in the absence of IRF4. These studies establish IRF4 as a transcriptional driver of a program of thermogenic gene expression and energy expenditure.

Effects of Wnt Signaling on Brown Adipocyte Differentiation and Metabolism Mediated by PGC-1α

ABSTRACT Activation of canonical Wnt signaling inhibits brown adipogenesis of cultured cells by impeding induction of PPARγ and C/EBPα. Although enforced expression of these adipogenic transcription factors restores lipid accumulation and expression of FABP4 in Wnt-expressing cells, additional expression of PGC-1α is required for activation of uncoupling protein 1 (UCP1). Wnt10b blocks brown adipose tissue development and expression of UCP1 when expressed from the fatty acid binding protein 4 promoter, even when mice are administered a β3-agonist. In differentiated brown adipocytes, activation of Wnt signaling suppresses expression of UCP1 through repression of PGC-1α. Consistent with these in vitro observations, UCP1-Wnt10b transgenic mice, which express Wnt10b in interscapular tissue, lack functional brown adipose tissue. While interscapular tissue of UCP1-Wnt10b mice lacks expression of PGC-1α and UCP1, the presence of unilocular lipid droplets and expression of white adipocyte genes suggest conversion of brown adipose tissue to white. Reciprocal expression of Wnt10b with UCP1 and PGC-1α in interscapular tissue from cold-challenged or genetically obese mice provides further evidence for regulation of brown adipocyte metabolism by Wnt signaling. Taken together, these data suggest that activation of canonical Wnt signaling early in differentiation blocks brown adipogenesis, whereas activating Wnt signaling in mature brown adipocytes stimulates their conversion to white adipocytes.

Regulation of early adipose commitment by Zfp521.

Zfp521 is a novel antiadipogenic transcription factor that helps to determine the identity of a mesenchymal cell as bone or fat.

Mammalian Stem Cells Reprogramming in Response to Terahertz Radiation

We report that extended exposure to broad-spectrum terahertz radiation results in specific changes in cellular functions that are closely related to DNA-directed gene transcription. Our gene chip survey of gene expression shows that whereas 89% of the protein coding genes in mouse stem cells do not respond to the applied terahertz radiation, certain genes are activated, while other are repressed. RT-PCR experiments with selected gene probes corresponding to transcripts in the three groups of genes detail the gene specific effect. The response was not only gene specific but also irradiation conditions dependent. Our findings suggest that the applied terahertz irradiation accelerates cell differentiation toward adipose phenotype by activating the transcription factor peroxisome proliferator-activated receptor gamma (PPARG). Finally, our molecular dynamics computer simulations indicate that the local breathing dynamics of the PPARG promoter DNA coincides with the gene specific response to the THz radiation. We propose that THz radiation is a potential tool for cellular reprogramming.

Inhibitor of DNA Binding 2 Is a Small Molecule-Inducible Modulator of Peroxisome Proliferator-Activated Receptor-γ Expression and Adipocyte Differentiation

We previously identified the small molecule harmine as a regulator of peroxisome proliferator activated-receptor gamma (PPARgamma) and adipocyte differentiation. In an effort to identify signaling pathways mediating harmines effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. Inhibitor of DNA biding 2 (Id2) was identified as a gene rapidly induced by harmine but not by PPARgamma agonists. Id2 is also induced in 3T3-L1 preadipocytes treated with dexamethasone, 3-isobutyl-1-methylxanthine, and insulin, suggesting that Id2 regulation is a common feature of the adipogenic program. Stable overexpression of Id2 in preadipocytes promotes expression of PPARgamma and enhances morphological differentiation and lipid accumulation. Conversely, small interfering RNA-mediated knockdown of Id2 antagonizes adipocyte differentiation. Mice lacking Id2 expression display reduced adiposity, and embryonic fibroblasts derived from these mice exhibit reduced PPARgamma expression and a diminished capacity for adipocyte differentiation. Finally, Id2 expression is elevated in adipose tissues of obese mice and humans. These results outline a role for Id2 in the modulation of PPARgamma expression and adipogenesis and underscore the utility of adipogenic small molecules as tools to dissect adipocyte biology.

Interferon Regulatory Factor 4 Regulates Obesity-Induced Inflammation Through Regulation of Adipose Tissue Macrophage Polarization

Interferon regulatory factors (IRFs) play functionally diverse roles in the transcriptional regulation of the immune system. We have previously shown that several IRFs are regulators of adipogenesis and that IRF4 is a critical transcriptional regulator of adipocyte lipid handling. However, the functional role of IRF4 in adipose tissue macrophages (ATMs) remains unclear, despite high expression there. Here we show that IRF4 expression is regulated in primary macrophages and in ATMs of high-fat diet–induced obese mice. Irf4−/− macrophages produce higher levels of proinflammatory cytokines, including interleukin-1β and tumor necrosis factor-α, in response to fatty acids. In coculture experiments, IRF4 deletion in macrophages leads to reduced insulin signaling and glucose uptake in 3T3-L1 adipocytes. To determine the macrophage-specific function of IRF4 in the context of obesity, we generated myeloid cell–specific IRF4 knockout mice, which develop significant insulin resistance on a high-fat diet, despite no difference in adiposity. This phenotype is associated with increased expression of inflammatory genes and decreased insulin signaling in adipose tissue, skeletal muscle, and liver. Furthermore, Irf4−/− ATMs express markers suggestive of enhanced M1 polarization. These findings indicate that IRF4 is a negative regulator of inflammation in diet-induced obesity, in part through regulation of macrophage polarization.

Retinoid-mediated inhibition of interleukin-12 production in mouse macrophages suppresses Th1 cytokine profile in CD4(+) T cells.

Interleukin‐12 (IL‐12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN‐γ and low IL‐4 production. In this study we investigated whether retinoid‐mediated inhibition of interleukin‐12 production in mouse macrophages could regulate cytokine profile of antigen (Ag)‐primed CD4+ Th cells. Pretreatment with retinoids (9‐cis‐RA, all‐trans‐RA, TTNPB) significantly inhibited IL‐12 production by mouse macrophages stimulated with lipopolysaccharide (LPS) or heated‐killed Listeria monocytogenes (HKL). Retinoid‐pretreated macrophages reduced their ability to induce IFN‐γ and increased the ability to induce IL‐4 in Ag‐primed CD4+ T cells. Addition of recombinant IL‐12 to cultures of retinoid‐pretreated macrophages and CD4+ T cells restored IFN‐γ production in CD4+ T cells. The in vivo administration of 9‐cis‐RA resulted in the inhibition of IL‐12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN‐γ and increased IL‐4 production) in CD4+ T cells. These findings may explain some known effects of retinoids including the inhibition of encephalitogenicity, and point to a possible therapeutic use of retinoids in the Th1‐mediated immune diseases such as autoimmune diseases.