Nahid Hemati

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.

Mechanisms of Drug-induced Lupus II. T Cells Overexpressing Lymphocyte Function-associated Antigen 1 Become Autoreactive and Cause a Lupuslike Disease in Syngeneic Mice

Current theories propose that systemic lupus erythematosus develops when genetically predisposed individuals are exposed to certain environmental agents, although how these agents trigger lupus is uncertain. Some of these agents, such as procainamide, hydralazine, and UV-light inhibit T cell DNA methylation, increase lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) expression, and induce autoreactivity in vitro, and adoptive transfer of T cells that are made autoreactive by this mechanism causes a lupuslike disease. The mechanism by which these cells cause autoimmunity is unknown. In this report, we present evidence that LFA-1 overexpression is sufficient to induce autoimmunity. LFA-1 overexpression was induced on cloned murine Th2 cells by transfection, resulting in autoreactivity. Adoptive transfer of the transfected, autoreactive cells into syngeneic recipients caused a lupuslike disease with anti-DNA antibodies, an immune complex glomerulonephritis and pulmonary alveolitis, similar to that caused by cells treated with procainamide. These results indicate that agents or events which modify T cell DNA methylation may induce autoimmunity by causing T cell LFA-1 overexpression. Since T cells from patients with active lupus have hypomethylated DNA and overexpressed LFA-1, this mechanism could be important in the development of human autoimmunity.

Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-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 β-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 β-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 β-catenin are also investigated. In conclusion, this study identifies Wnt10a and Wnt6 as additional regulators of MSC fate and demonstrates that mechanisms downstream of β-catenin are required for Wnt6, Wnt10a and Wnt10b to influence differentiation of mesenchymal precursors.

Glycogen Synthase Kinase 3 Is an Insulin-Regulated C/EBPα Kinase

ABSTRACT CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor involved in creating and maintaining the adipocyte phenotype. We have shown previously that insulin stimulates dephosphorylation of C/EBPα in 3T3-L1 adipocytes. Studies to identify the insulin-sensitive sites of phosphorylation reveal that a C/EBPα peptide (amino acids H215 to K250) is phosphorylated on T222, T226, and S230 in vivo. The context of these phosphoamino acids implicates glycogen synthase kinase 3 (GSK3), whose activity is known to be repressed in response to insulin, as a potential kinase for phosphorylation of T222 and T226. Accordingly, GSK3 phosphorylates the predicted region of C/EBPα on threonine in vitro, and GSK3 uses C/EBPα as a substrate in vivo. In addition, the effect of pharmacological agents on GSK3 activity correlates with regulation of C/EBPα phosphorylation. Treatment of 3T3-L1 adipocytes with the phosphatidylinositol 3-kinase inhibitor wortmannin results in phosphorylation of C/EBPα, whereas treatment with the GSK3 inhibitor lithium results in dephosphorylation of C/EBPα. Collectively, these data indicate that insulin stimulates dephosphorylation of C/EBPα on T222 and T226 through inactivation of GSK3. Since dephosphorylation of C/EBPα in response to lithium is blocked by okadaic acid, strong candidates for the T222 and T226 phosphatase are protein phosphatases 1 and 2a. Treatment of adipocytes with insulin alters the protease accessibility of widespread sites within the N terminus of C/EBPα, consistent with phosphorylation causing profound conformational changes. Finally, phosphorylation of C/EBPα and other substrates by GSK3 may be required for adipogenesis, since treatment of differentiating preadipocytes with lithium inhibits their conversion to adipocytes.

Signaling Pathways through Which Insulin Regulates CCAAT/Enhancer Binding Protein α (C/EBPα) Phosphorylation and Gene Expression in 3T3-L1 Adipocytes CORRELATION WITH GLUT4 GENE EXPRESSION

Treatment of 3T3-L1 adipocytes with insulin (IC50 ∼200 pm insulin) or insulin-like growth factor-1 (IC50 ∼200 pm IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein α (C/EBPα), a transcription factor involved in preadipocyte differentiation. As assessed by immunoblot analysis of one- and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBPα and an additional site within p42C/EBPα. Consistent with insulin causing dephosphorylation of C/EBPα through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPα. In the absence of insulin, wortmannin or LY294002 enhance C/EBPα phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPα in the absence of insulin. Dephosphorylation of C/EBPα by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBPα. The dephosphorylation of C/EBPα by insulin, in conjunction with the insulin-dependent decline in C/EBPα mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBPα. However, the repression of C/EBPα mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBPα and GLUT4 mRNAs by insulin. A decline in C/EBPα mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPβ (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.

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

Background: Paired-related homeobox 1 (Prrx1) regulates mesenchymal cell fate, but whether Prrx1 impacts adipogenesis remains unknown. Results: Prrx1 knockdown decreases transforming growth factor-β (TGFβ) ligand expression and enhances adipogenesis, whereas Prrx1 increases in adipose tissue during obesity. Conclusion: Prrx1 knockdown enhances adipogenesis by suppressing TGFβ signaling. Significance: This report identifies Prrx1 as an inhibitor of adipogenesis that may impact adipose tissue function in obesity. 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 TGFβ signaling during adipogenesis mimics the effects of Prrx1 knockdown. These data support the hypothesis that endogenous Prrx1 restrains adipogenesis by regulating expression of TGFβ ligands and thereby activating TGFβ 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 TGFβ activity in adipose tissue and thereby contribute to aberrant adipocyte function during obesity.