The transcription factor paired-related homeobox 1 (Prrx1) inhibits adipogenesis by activating transforming growth factor-ß (TGFß) signaling.

Differentiation of adipocytes from preadipocytes contributes to adipose tissue expansion in obesity. Impaired adipogenesis may underlie the development of metabolic diseases such as insulin resistance and type 2 diabetes. Mechanistically, a well defined transcriptional network coordinates adipocyte differentiation. The family of paired-related homeobox transcription factors, which includes Prrx1a, Prrx1b, and Prrx2, is implicated with regulation of mesenchymal cell fate, including myogenesis and skeletogenesis; however, whether these proteins impact adipogenesis remains to be addressed. In this study, we identify Prrx1a and Prrx1b as negative regulators of adipogenesis. We show that Prrx1a and Prrx1b are down-regulated during adipogenesis in vitro and in vivo. Stable knockdown of Prrx1a/b enhances adipogenesis, with increased expression of peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α and FABP4 and increased secretion of the adipokines adiponectin and chemerin. Although stable low-level expression of Prrx1a, Prrx1b, or Prrx2 does not affect 3T3-L1 adipogenesis, transient overexpression of Prrx1a or Prrx1b inhibits peroxisome proliferator-activated receptor-γ activity. Prrx1 knockdown decreases expression of Tgfb2 and Tgfb3, and inhibition of TGFß signaling during adipogenesis mimics the effects of Prrx1 knockdown. These data support the hypothesis that endogenous Prrx1 restrains adipogenesis by regulating expression of TGFß ligands and thereby activating TGFß signaling. Finally, we find that expression of Prrx1a or Prrx1b in adipose tissue increases during obesity and strongly correlates with Tgfb3 expression in BL6 mice. These observations suggest that increased Prrx1 expression may promote TGFß activity in adipose tissue and thereby contribute to aberrant adipocyte function during obesity.

Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a ß-catenin-dependent mechanism.

Wnt10b is an established regulator of mesenchymal stem cell (MSC) fate that inhibits adipogenesis and stimulates osteoblastogenesis, thereby impacting bone mass in vivo. However, downstream mechanisms through which Wnt10b exerts these effects are poorly understood. Moreover, whether other endogenous Wnt ligands also modulate MSC fate remains to be fully addressed. In this study, we identify Wnt6 and Wnt10a as additional Wnt family members that, like Wnt10b, are downregulated during development of white adipocytes in vivo and in vitro, suggesting that Wnt6 and/or Wnt10a may also inhibit adipogenesis. To assess the relative activities of Wnt6, Wnt10a and Wnt10b to regulate mesenchymal cell fate, we used gain- and loss-of function approaches in bipotential ST2 cells and in 3T3-L1 preadipocytes. Enforced expression of Wnt10a stabilizes ß-catenin, suppresses adipogenesis and stimulates osteoblastogenesis to a similar extent as Wnt10b, whereas stable expression of Wnt6 has a weaker effect on these processes than Wnt10a or Wnt10b. In contrast, knockdown of endogenous Wnt6 is associated with greater preadipocyte differentiation and impaired osteoblastogenesis than knockdown of Wnt10a or Wnt10b, suggesting that, among these Wnt ligands, Wnt6 is the most potent endogenous regulator of MSC fate. Finally, we show that knockdown of ß-catenin completely prevents the inhibition of adipogenesis and stimulation of osteoblast differentiation by Wnt6, Wnt10a or Wnt10b. Potential mechanisms whereby Wnts regulate fate of MSCs downstream of ß-catenin are also investigated. In conclusion, this study identifies Wnt10a and Wnt6 as additional regulators of MSC fate and demonstrates that mechanisms downstream of ß-catenin are required for Wnt6, Wnt10a and Wnt10b to influence differentiation of mesenchymal precursors.

Centrosome misorientation reduces stem cell division during ageing.

Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. Here we show that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. We found that GSCs containing misoriented centrosomes accumulate with age and that these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, we propose that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. We also show that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia.

RPE65 surface epitopes, protein interactions, and expression in rod- and cone-dominant species.

PURPOSE: RPE65 is an abundant protein necessary for the synthesis of the chromophore 11-cis retinal by the retinal pigment epithelium (RPE). Our purpose was to identify RPE65 surface epitopes, to assess protein interactions, and to evaluate RPE65 expression in eyes from rod- and cone-dominant species using a monoclonal antibody approach. METHODS: RPE65-specific monoclonal antibodies, mAb 8B11, and mAb 1F9, were generated using bovine RPE microsomal membranes and a human RPE65 synthetic peptide as immunogen, respectively. Western analysis was performed on bovine RPE membranes, as well as yeast strains generated by transfection with RPE65 cDNAs. Competition of antibody binding by synthetic peptides was assayed using ELISAs, western analysis, and elution from immunoaffinity matrices. RPE65 structural models were generated by ab initio and comparative methods. Immunohistochemistry was performed on retina/RPE/choroid cryosections and retina flatmounts. RESULTS: The antigenic determinant recognized by mAb 8B11 was localized to a 10 amino acid sequence, KVNPETLETI, that competed binding with microM affinity and eluted RPE65 from an immunoaffinity matrix incubated with solubilized bovine RPE membranes or RPE65-transfected cells. Similarly, solubilized RPE65 was bound and eluted from an mAb 1F9 immunoaffinity matrix using the immunizing peptide, FHHINTYEDNGFLIV. In both cases, 11-cis retinol dehydrogenase, but not other known visual cycle proteins, appeared to co-elute with RPE65 in substoichiometric amounts. Both sequences localized to surface exposed regions of predicted RPE65 tertiary structures. RPE65 immunoreactivity was detected by mAb 8B11 and mAb 1F9 in the RPE, but not in retina, in bovine, rat, mouse, human, chicken, and Xenopus laevis, and in Nrl knockout mice whose retinas contain exclusively cone-like photoreceptor cells. CONCLUSIONS: The identification of RPE65 surface exposed antigenic determinants represents a first step toward understanding RPE65 structure and its interaction with visual cycle proteins, and provides a means for the purification of the native protein. The finding that RPE65 immunoreactivity is present in the RPE and not retina of both rod- and cone-dominant species does not support a proposed direct role for RPE65 in cone cell function.

Regulation of Wnt signaling during adipogenesis.

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.