STAT Protein Recruitment and Activation in c-Kit Deletion Mutants

Stem cell factor (SCF) and its tyrosine kinase receptor, c-Kit, play a crucial role in regulating migration and proliferation of melanoblasts, germ cells, and hemopoietic cell progenitors by activating a number of intracellular signaling molecules. Here we report that SCF stimulation of myeloid cells or fibroblasts ectopically expressing c-Kit induces physical association with and tyrosine phosphorylation of three signal transducers and activators of transcription (STATs) as follows: STAT1alpha, STAT5A, and STAT5B. Other STAT proteins are not recruited upon SCF stimulation. Recruitment of STATs leads to their dimerization, nuclear translocation, and binding to specific promoter-responsive elements. Whereas STAT1alpha, possibly in the form of homodimers, binds to the sis-inducible DNA element, STAT5 proteins, either as STAT5A/STAT5B or STAT5/STAT1alpha heterodimers, bind to the prolactin-inducible element of the beta-casein promoter. The tyrosine kinase activity of Kit appears essential for STAT activation since a kinase-defective mutant lacking a kinase insert domain was inactive in STAT signaling. However, another mutant that lacked the carboxyl-terminal region retained STAT1alpha activation and nuclear translocation but was unable to fully activate STAT5 proteins, although it mediated their transient phosphorylation. These results indicate that different intracellular domains of c-Kit are involved in activation of the various STAT proteins

microRNA-222 controls neovascularization by regulating signal transducer and activator of transcription 5A expression.

OBJECTIVE: Inflammatory stimuli released into atherosclerotic plaque microenvironment regulate vessel formation by modulating gene expression and translation. microRNAs are a class of short noncoding RNAs, acting as posttranscriptional regulators of protein-coding genes involved in various biological processes, including vascular cell biology. Among them, microRNA-221/222 (miR-221/222) seem to negatively modulate vascular remodeling by targeting different target genes. Here, we investigated their potential contribution to inflammation-mediated neovessel formation. METHODS AND RESULTS: We used quantitative real-time RT-PCR amplification to analyze expression of 7 microRNAs previously linked to vascular biology, such as miR-17-5p, miR-21, miR-126, miR-210, miR-221, miR-222, and miR-296 and found high levels of expression for all of them in quiescent endothelial cells. However, miR-126, miR-221, miR-222, and miR-296 turned out to be down-modulated in endothelial cells exposed to inflammatory stimuli. Applying a gain-of-function approach, we demonstrated that, among them, only miR-222 was involved in inflammation-mediated vascular remodeling. In addition, we identified signal transducer and activator of transcription 5A (STAT5A) as a bona fide target of miR-222 and observed that miR-222 negatively correlated with STAT5A expression in human endothelial cells from advanced neovascularized atherosclerotic lesions. CONCLUSIONS: We identified STAT5A as a novel miR-222 target, and this finding opens up new perspectives for treatment of vascular diseases

Oxidative Stress-mediated Mesangial Cell Proliferation Requires RAC-1/Reactive Oxygen Species Production and β4 Integrin Expression

Abstract Lipid abnormalities and oxidative stress, by stimulating mesangial cell (MC) proliferation, can contribute to the development of diabetes-associated renal disease. In this study we investigated the molecular events elicited by oxidized low density lipoproteins (ox-LDL) in MC. We demonstrate that in MC cultured in the presence of ox-LDL, survival and mitogenic signals on Akt and Erk1/2 MAPK pathways are induced, respectively. Moreover, as shown by the expression of the dominant negative Rac-1 construct, we first report that ox-LDL-mediated cell survival and cell cycle progression depend on Rac-1 GTPase-mediated reactive oxygen species production and on epidermal growth factor receptor transactivation. By silencing Akt and blocking Erk1/2 MAPK pathways, we also demonstrate that these signals are downstream to Rac-1/reactive oxygen species production and epidermal growth factor receptor activation. Finally, by endogenous depletion of β4 integrin, expressed in MC, we provide evidence that the expression of this adhesion molecule is essential for ox-LDL-mediated MC dysfunction. Our data identify a novel signaling pathway involved in oxidative stress-induced diabetes-associated renal disease and provide the rationale for therapeutically targeting β4 integrin

p53 Mediates the Accelerated Onset of Senescence of Endothelial Progenitor Cells in Diabetes

Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes

β1 integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation

We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3–mediated signaling. Here, we show that in endothelial cells the active β1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) β common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the β1 integrin–IL-3R complex and triggers IL-3R β common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R β common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3–mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3–mediated proliferation

Unacylated Ghrelin Promotes Skeletal Muscle Regeneration Following Hindlimb Ischemia via SOD-2-Mediated miR-221/222 Expression

BACKGROUND: Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated‐ghrelin (UnAG) to reduce ischemia‐induced tissue damage in a mouse model of peripheral artery disease. METHODS AND RESULTS: We show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen‐actived protein kinase signaling UnAG protected against reactive oxygen species–induced cell injuries by inducing the expression of superoxide dismutase‐2 (SOD‐2) in satellite cells. This led to a reduced number of infiltrating CD68(+) cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR‐221/222, previously linked to muscle regeneration processes, was up‐regulated and negatively correlated with p57(Kip2) expression in UnAG‐treated mice. UnAG, unlike AG, promoted cell‐cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG‐induced p38/mitogen‐actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD‐2–depleted SCs. By siRNA technology, we also demonstrated that SOD‐2 is the antioxidant enzyme involved in the control of miR‐221/222–driven posttranscriptional p57(Kip2) regulation. Loss‐of‐function experiments targeting miR‐221/222 and local pre–miR‐221/222 injection in vivo confirmed a role for miR‐221/222 in driving skeletal muscle regeneration after ischemia. CONCLUSIONS: These results indicate that UnAG‐induced skeletal muscle regeneration after ischemia depends on SOD‐2–induced miR‐221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species–mediated skeletal muscle damage

Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action.

Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices