Regulation of secreted protein acidic and rich in cysteine during adipose conversion and adipose tissue hyperplasia.

OBJECTIVE: To explore the regulation of secreted protein acidic and rich in cysteine (SPARC) expression and its role in adipose tissue. RESEARCH METHODS AND PROCEDURES: We studied the regulation of SPARC expression in transgenic mice expressing the human beta3 and alpha2 adrenergic receptors on a murine beta3 adrenergic receptor null background that became obese under a high-fat diet mainly as a result of adipose tissue hyperplasia. Furthermore, we analyzed its expression in human adipose tissue and its regulation during adipocyte differentiation. RESULTS: SPARC protein in adipose tissue was increased in obese transgenic mice compared with control mice, indicating that SPARC expression was associated with adipose tissue hyperplasia. Both SPARC mRNA and protein were detected in human adipose tissue. Comparing adipocytes and vascular stroma, we found that SPARC expression was mainly associated with the adipocyte fraction. Consistent with this, SPARC transcript increased during differentiation of human primary preadipocytes. 3T3-L1 preadipocytes showed an increase in SPARC expression in differentiated cells but with biphasic expression during the process. After induction in committed cells, SPARC mRNA and protein levels declined as differentiation began and returned to elevated levels in fully differentiated adipocytes. DISCUSSION: SPARC expression correlated with adipose tissue hyperplasia and adipogenesis. Therefore, SPARC seems to play a role in adipose tissue physiology as it is involved in growth and differentiation.

Regulation of hypoxia-inducible factor (HIF)-1 activity and expression of HIF hydroxylases in response to insulin-like growth factor I.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of two subunits (HIF-1alpha and HIF-1beta), initially described as a mediator of adaptive responses to changes in tissue oxygenation, has been shown to be activated in an oxygen-independent manner. In this report, we studied the action of IGF-I on the regulation of HIF-1 in human retinal epithelial cells. We show that IGF-I stimulates HIF-1alpha accumulation, HIF-1alpha nuclear translocation, and HIF-1 activity by regulation of HIF-1alpha expression through a posttranscriptional mechanism. In addition, we demonstrate that IGF-I stimulates HIF-1 activity through phosphatidylinositol-3-kinase/ mammalian target of rapamycin and MAPK-dependent signaling pathways leading to VEGF (vascular endothelial growth factor) mRNA expression. Three human prolyl-hydroxylases PHD-1, -2, and -3 (PHD-containing protein) and an asparaginyl-hydroxylase factor inhibiting HIF-1, which regulate HIF-1alpha stability and HIF-1 activity in response to hypoxia, have been described. Our analysis of their mRNA expression showed a different magnitude and time course of expression pattern in response to insulin and IGF-I compared with CoCl(2). Taken together, our data reveal that growth factors and CoCl(2), which mimics hypoxia, lead to HIF-1 activation and ensuing VEGF expression by different mechanisms. Their joined actions are likely to lead to an important and sustained increase in VEGF action on retinal blood vessels, and hence to have devastating effects on the development of diabetic retinopathy.

Grb10 prevents Nedd4-mediated vascular endothelial growth factor receptor-2 degradation.

One of the cellular mechanisms used to prevent continuous and enhanced activation in response to growth factors is the internalization and degradation of their receptors. Little is known about the molecular mechanisms involved in vascular endothelial growth factor receptor-2 (VEGF-R2) degradation. In a previous work, we have shown that the adaptor protein Grb10 is a positive regulator of the VEGF signaling pathway. Indeed, VEGF stimulates Grb10 expression, and Grb10 overexpression induces an increase in the amount and the tyrosine phosphorylation of VEGF-R2. In the present manuscript, we demonstrate that Grb10 stimulates VEGF-R2 expression by inhibiting the Nedd4-mediated VEGF-R2 degradation. First, we show that proteasome inhibition by MG132 induces an increase in VEGF-R2 amount, and that VEGF-R2 is ubiquitinated in response to VEGF. Expression of Nedd4, a HECT domain-containing ubiquitin ligase, induces the disappearance of VEGF-R2 in cells, suggesting that Nedd4 is involved in VEGF-R2 degradation. To determine whether Nedd4 directly ubiquitinates VEGF-R2, we expressed a ubiquitin ligase-deficient mutant Nedd4C854S. In the presence of Nedd4C854S, VEGF-R2 is expressed and ubiquitinated. These results suggest that VEGF-R2 is ubiquitinated but that Nedd4 is not involved in this process. Finally, we show that Grb10 constitutively associates with Nedd4. Co-expression of Nedd4 and Grb10 restores the expression of VEGF-R2, suggesting that Grb10 inhibits the Nedd4-mediated degradation of VEGF-R2. In this study, we show that Grb10 acts as a positive regulator in VEGF-R2 signaling and protects VEGF-R2 from degradation by interacting with Nedd4, a component of the endocytic machinery.