Treatment with XMU-MP-1 erases hyperglycaemic memory in hearts of diabetic mice.

Hyperglycaemic memory refers to the damages occurred under early hyperglycaemic environment in organs of diabetic patients persisting after intensive glycaemic control. Mammalian sterile 20-like kinase 1 (Mst1) contributes to the development of diabetic cardiomyopathy. Here, we investigated the role of Mst1 in hyperglycaemic memory and test the effect of XMU-MP-1, a Mst1 inhibitor, on hyperglycaemic memory in hearts. Eight weeks after induction of type 1 diabetes by injection with streptozotocin (STZ) in mice, glycaemic control was obtained by means of insulin treatment and maintained for 4 additional weeks. In the diabetic mice, insulin treatment alone did not reduce phosphorylation of Mst1 or improve cardiac function. Treatment with XMU-MP-1 alone immediately after induction of diabetes for 12 weeks did not improve myocardial function in mice. But treatment with XMU-MP-1 for the later 4 weeks relieved myocardial dysfunction when glycaemic control was obtained by insulin treatment simultaneously. Mst1 deficiency and glycaemic control synergistically improved myocardial function and reduced apoptosis in myocardium of diabetic mice. Mechanistically, when Mst1 was deficient or inhibited by XMU-MP-1, AMPK was activated and mitochondrial dysfunction was attenuated. In vitro, treatment with AMPK activator reversed the detrimental effects of Mst1 overexpression in cultured cardiomyocytes. XMU-MP-1 might thus be envisaged as a complement for insulin treatment against diabetic cardiomyopathy.

MST1 deficiency promotes B cell responses by CD4+ T cell-derived IL-4, resulting in hypergammaglobulinemia.

MST1 deficiency causes T and B cell lymphopenia, resulting in combined immunodeficiency. However, MST1-deficient patients also exhibit autoimmune-like symptoms such as hypergammaglobulinemia and autoantibody production. Recent studies have shown that the autoimmune responses observed in MST1-deficient patients were most likely attributable to defective regulatory T (Treg) cells instead of intrinsic signals in MST1-lacking B cells. Nevertheless, it is not determined how MST1 deficiency in T cells breaks B cell tolerance and causes systemic autoimmune-like phenotypes. In this study, we confirmed that Mst1-/- mice developed hypergammaglobulinemia associated with increased levels of IgG, IgA, and IgE. We also showed that uncontrolled B cell responses were resulted from the IL-4-rich environment created by CD4+ T cells. Defective MST1-FOXO1 signaling down-regulated Treg cells, resulting in the collapse of immune tolerance where the populations of Th2 and T follicular helper cells expanded. In conclusion, we suggest that MST1 acts as a molecular brake to maintain immune tolerance by regulating T cell-mediated B cell activation.

Genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1) improves cell reprogramming efficiency and increases induced pluripotent stem cell proliferation and survival.

Adult fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) for use in various applications. However, there are challenges in iPSC generation including low reprogramming efficiency, yield, cell survival and viability. Since the Hippo signalling pathway is a key pathway involved in regulating cell proliferation and survival, we here test whether modification of the Hippo pathway will enhance the efficiency of iPSC generation and improve their survival. The Hippo pathway was modified by genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1), a major component of the pathway. Using adult skin fibroblasts isolated from Mst1 knockout mice (Mst1-/-) as a source of iPSC we found that genetic ablation of Mst1 leads to significantly increased reprogramming efficiency by 43.8%. Moreover, Mst1-/- iPSC displayed increase proliferation by 12% as well as an increase in cell viability by 20% when treated with a chemical hypoxic inducer. Mechanistically, we found higher activity of YAP, the main downstream effector of the Hippo pathway, in iPSC lacking Mst1. In conclusion, our data suggests that Mst1 can be targeted to improve the efficiency of adult somatic cell reprogramming as well as to enhance iPSC proliferation and survival.

Mst1 participates in the atherosclerosis progression through macrophage autophagy inhibition and macrophage apoptosis enhancement.

Emerging evidence favors the notion that macrophage autophagy plays a prominent role in the pathogenesis of vulnerable plaque, suggesting the therapeutic potential of targeting autophagy in atherosclerosis. Here ApoE(-/-) mice were crossed with Mst1 knockout or Mst1 Tg mice to generate ApoE(-/-):Mst1(-/-) and ApoE(-/-):Mst1Tg mice. All animals were fed high-fat-diet for 4months to induce arterial atherosclerosis. Murine macrophage RAW264.7 cells were subjected to ox-LDL (50µg/mL) in an effort to examine the cellular mechanisms. A significant increase in the levels of Mst1 and p-Mst1 was observed in the aorta of ApoE(-/-) mice. Mst1 knockout significantly reduced atherosclerotic area, decreased lipid core area and macrophage accumulation as compared with ApoE(-/-) mice. Along the same line, Mst1 overexpression increased plaque area, lipid core and macrophage accumulation as compared with ApoE(-/-) mice. Mst1 deficiency significantly increased levels of Beclin1 and LC3II, while decreased that of p62 in aortic atherosclerosis. Moreover, in vitro data indicated that Mst1 knockdown prompted more typical autophagosomes upon ox-LDL challenge. Mst1 knockdown also enhanced autophagic flux as evidenced by GFP-mRFP-LC3 staining, increased LC3-II expression and decreased p62 expression in the presence of bafilomycin A1. Mst1 knockdown decreased, while Mst1 overexpression increased macrophage apoptosis upon ox-LDL exposure. In conclusion, Mst1 deficiency diminishes atherosclerosis and stabilizes atherosclerotic plaques in ApoE(-/-) mice. Mst1 may participate in atherosclerosis progression through inhibition of macrophage autophagy and promotion of macrophage apoptosis.

Deficiency in macrophage-stimulating protein results in spontaneous intestinal inflammation and increased susceptibility toward epithelial damage in zebrafish.

Several genome-wide association studies have identified the genes encoding for macrophage-stimulating protein (MSP) and its receptor RON (Recepteur d'Origine Nantais) as possible susceptibility factors in inflammatory bowel disease. While it has been shown that the MSP-RON signaling pathway is involved in tissue injury responses, current mouse models for MSP and RON deficiency have not clearly demonstrated a role of MSP-RON signaling in the context of intestinal inflammation. In this study, we report that the recently identified zebrafish Msp mutant (msp(t34230)) develops spontaneous intestinal inflammation over time. From 14 to 28 weeks postfertilization Msp-deficient zebrafish show intestinal eosinophilia, increased intestinal expression of inflammatory marker mmp9, and activation of intestinal goblet cells. Moreover, these Msp mutant zebrafish are more susceptible toward ethanol-induced epithelial damage, which resulted in increased infiltration and proliferation of immune cells within the lamina propria and prolonged intestinal proinflammatory cytokine responses in some mutant fish. In light of the recent development of many tools to visualize, monitor, and genetically modify zebrafish, these Msp-deficient zebrafish will enable in-depth in vivo analysis of epithelial and macrophage-specific MSP-RON signaling in the context of intestinal inflammation.

Hepatocyte growth factor-like protein is required for prostate tumor growth in the TRAMP mouse model.

The Ron receptor is deregulated in a variety of cancers. Hepatocyte growth factor-like protein (HGFL) is the ligand for Ron and is constitutively secreted from hepatocytes into the circulation. While a few recent reports have emerged analyzing ectopic HGFL overexpression of in cancer cells, no studies have examined host-produced HGFL in tumorigenesis. To examine HGFL function in prostate cancer, the TRAMP mouse model, which is predisposed to develop prostate tumors, was utilized. Prostate tumors from TRAMP mice exhibit elevated levels of HGFL, which correlated with upregulation in human prostate cancer. To directly implicate HGFL in prostate tumorigenesis, TRAMP mice deficient in HGFL (HGFL-/-TRAMP+) were generated. HGFL-/- TRAMP+ mice developed significantly smaller prostate tumors compared to controls. Analysis of HGFL-/- tumors revealed reduced tumor vascularization. No differences in cancer cell proliferation were detected between HGFL-/- TRAMP+ and HGFL+/+ TRAMP+ mice. However, a significant increase in cancer cell death was detected in HGFL-/- TRAMP+ prostates which correlated with decreased pro-survival targets. In vitro analysis demonstrated robust STAT3 activation resulting in Bcl2-dependent survival following treatment of prostate cancer cells with HGFL. These data document a novel function for endogenous HGFL in prostate cancer by imparting a critical survival signal to tumor cells.

Hepatocyte growth factor-like protein is a positive regulator of early mammary gland ductal morphogenesis.

The Ron receptor tyrosine kinase regulates multiple cellular processes and is important during mammary gland development and tumor progression. Hepatocyte growth factor-like protein [HGFL] is the only known ligand for the Ron receptor and recent studies have identified major roles for HGFL during breast cancer metastasis. Understanding the functional importance HGFL during mammary gland development will provide significant insights onto its contribution during tumor development and metastasis. In this study, we assessed the role of HGFL during postnatal mammary gland development using mice that were either proficient [HGFL +/+] or deficient [HGFL-/-] for HGFL. Postnatal ductal morphology and stromal cell associations were analyzed at multiple time points through puberty until adulthood. HGFL deficiency resulted in several mammary gland developmental defects including smaller terminal end buds [TEBs], significantly fewer TEBs, and delayed ductal outgrowth during early puberty. Additionally, HGFL deficient animals exhibited significantly altered TEB epithelial cell turnover with decreased proliferation and increased apoptosis coupled with decreased TEB diameter. Macrophage recruitment to the TEBs was also significantly decreased in the HGFL-/- mice compared to controls. Moreover, the levels of STAT3 mRNA as well as the phosphorylation status of this protein were lower in the HGFL-/- mammary glands compared to controls. Taken together, our data provide the first evidence for HGFL as a positive regulator of mammary gland ductal morphogenesis by controlling overall epithelial cell turnover, macrophage recruitment, and STAT3 activation in the developing mammary gland. With a function in early mammary gland development, HGFL represents a potential target for the development of novel breast cancer therapies.

Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice.

UNLABELLED: Sorafenib--a broad kinase inhibitor--is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia--and its consequences on treatment resistance--remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1α) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen-positive (Gr-1(+)) myeloid cell infiltration. The SDF-1α/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1α expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1(+) myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis--despite persistently elevated hypoxia--in part by reducing Gr-1(+) myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. CONCLUSION: Blocking SDF-1α/CXCR4 or Gr-1(+) myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment.

Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2.

Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.