Carmen Guerrero

Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

β-Agonists regulate Na,K-ATPase via novel MAPK/ERK and rapamycin-sensitive pathways

We studied whether the β‐adrenergic agonist, isoproterenol (ISO), regulates Na,K‐ATPase in alveolar epithelial cells (AEC) via a mitogen‐activated protein kinase (MAPK)/extracellular signaling related kinase (ERK) dependent pathway. ISO increased ERK activity in AEC by 10 min via a β‐adrenergic receptor, protein kinase A (PKA)‐dependent mechanism. Activation of the MAPK pathway by ISO, resulted in increased Na,K‐ATPase β1 and α1 subunit protein abundance in whole cell lysates, which resulted in functional Na,K‐ATPases at the basolateral membranes. ISO did not change the α1 or β1 mRNA steady state levels, but rapamycin, the inhibitor of the mammalian target of rapamycin, also blocked the ISO‐mediated increase in Na,K‐ATPase total protein abundance, suggesting a posttranscriptional regulation. We conclude that ISO, regulates the Na,K‐ATPase in AEC via PKA, ERK and rapamycin‐sensitive mechanisms.

Ras-Guanine Nucleotide Exchange Factor Sos2 Is Dispensable for Mouse Growth and Development

ABSTRACT The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function ofsos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2+/− mice produced viablesos2−/− offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2−/− mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1,sos2 gene function is dispensable for normal mouse development, growth, and fertility.

Transcriptional networks of knockout cell lines identify functional specificities of H-Ras and N-Ras: significant involvement of N-Ras in biotic and defense responses

We characterized differential gene expression profiles of fibroblast cell lines harboring single or double-homozygous null mutations in H-ras and N-ras. Whereas the expression level of the individual H-, N- and K-ras genes appeared unaffected by the presence or absence of the other ras loci, significant differences were observed between the expression profiles of cells missing N-ras and/or H-ras. Absence of N-ras produced much stronger effects than absence of H-ras over the profile of the cellular transcriptome. N-ras−/− and H-ras−/− fibroblasts displayed rather antagonistic expression profiles and the transcriptome of H-ras−/− cells was significantly closer to that of wild-type fibroblasts than to that of N-ras−/− cells. Classifying all differentially expressed genes into functional categories suggested specific roles for H-Ras and N-Ras. It was particularly striking in N-ras−/− cells the upregulation of a remarkable number of immunity-related genes, as well as of several loci involved in apoptosis. Reverse-phase protein array assays demonstrated in the same N-ras−/− cells the overexpression and nuclear migration of tyrosine phosphorylated signal transducer and activator of transcription 1 (Stat1) which was concomitant with transcriptional activation mediated by interferon-stimulated response elements. Significantly enhanced numbers of apoptotic cells were also detected in cultures of N-ras−/− cells. Our data support the notion that different Ras isoforms play functionally distinct cellular roles and indicate that N-Ras is significantly involved in immune modulation/host defense and apoptotic responses

C3G-mediated suppression of oncogene-induced focus formation in fibroblasts involves inhibition of ERK activation, cyclin A expression and alterations of anchorage-independent growth.

We showed previously that exogenous overexpression of C3G, a guanine nucleotide releasing factor (GEF) for Rap1 and R-Ras proteins, blocks the focus-forming activity of cotransfected, activated, sis, ras and v-raf oncogenes in NIH 3T3 cells. In this report, we show that C3G also interferes with dbl and R-Ras focus-forming activity and demonstrate that the transformation suppressor ability of C3G maps to its Crk-binding region (SH3-b domain). Using full-length C3G and C3GΔCat mutant, lacking catalytic domain, we showed here that overexpression of cotransfected C3G or C3GΔCat inhibited oncogenic Hraslys12-mediated phosphorylation of ERK, without altering Ras and Raf-1 kinase activation. We also showed that, overexpressed C3G and C3GΔCat inhibited the viability of oncogenic Ras-induced colonies in soft agar, indicating that C3G interferes with the anchorage-independent growth of Ras-transformed cells in a Rap1-independent manner. Consistent with both observations, overexpression of exogenous C3G and C3GΔCat also caused downregulation of Ras-induced cyclin A expression. Altogether, our results indicate that C3G interferes with at least two separate aspects of oncogenic transformation – cell cycle progression and loss of contact inhibition – and that these inhibitory effects probably account for its transformation suppressor activity.

Analysis of incidence, risk factors and clinical outcome of thromboembolic and bleeding events in 431 allogeneic hematopoietic stem cell transplantation recipients

Allogeneic hematopoietic stem cell transplantation recipients have an increasing risk of both hemorrhagic and thrombotic complications. However, the competing risks of two of these life-threatening complications in these complex patients have still not been well defined. We retrospectively analyzed data from 431 allogeneic transplantation recipients to identify the incidence, risk factors and mortality due to thrombosis and bleeding. Significant clinical bleeding was more frequent than symptomatic thrombosis. The cumulative incidence of a bleeding episode was 30.2% at 14 years. The cumulative incidence of a venous or arterial thrombosis at 14 years was 11.8% and 4.1%, respectively. The analysis of competing factors for venous thrombosis revealed extensive chronic graft-versus-host disease to be the only independent prognostic risk factor. By contrast, six factors were associated with an increased risk of bleeding; advanced disease, ablative conditioning regimen, umbilical cord blood transplantation, anticoagulation, acute III-IV graft-versus-host disease, and transplant-associated microangiopathy. The development of thrombosis did not significantly affect overall survival (P=0.856). However, significant clinical bleeding was associated with inferior survival (P<0.001). In allogeneic hematopoietic stem cell transplantation, significant clinical bleeding is more common than thrombotic complications and affects survival.

C3G down-regulates p38 MAPK activity in response to stress by Rap-1 independent mechanisms: involvement in cell death.

We present here evidences supporting a negative regulation of p38alpha MAPK activity by C3G in MEFs triggered by stress, which can mediate cell death or survival depending on the stimuli. Upon serum deprivation, C3G induces survival through inhibition of p38alpha activation, which mediates apoptosis. In contrast, in response to H2O2, C3G behaves as a pro-apoptotic molecule, as its knock-down or knock-out enhances survival through up-regulation of p38alpha activation, which plays an anti-apoptotic role under these conditions. Moreover, the C3G target, Rap-1, plays an opposite role, also through regulation of p38alpha MAPK activity. Our data also suggest that changes in the protein levels of some members of the Bcl-2 family could account for the regulation of cell death by C3G and/or Rap-1 through p38alpha MAPK. Bim/Bcl-xL ratio appears to be important in the regulation of cell survival, both upon serum deprivation and in response to H2O2. In addition, the increase in BNIP-3 levels induced by C3G knock-down in wt cells treated with H2O2 might play a role preventing cell death. Therefore, we can conclude that C3G is a negative regulator of p38alpha MAPK in MEFs, while Rap-1 is a positive regulator, but both, through the regulation of p38alpha activity, can promote cell survival or cell death depending on the stimuli.

Met signaling in cardiomyocytes is required for normal cardiac function in adult mice

Hepatocyte growth factor (HGF) and its receptor, Met, are key determinants of distinct developmental processes. Although HGF exerts cardio-protective effects in a number of cardiac pathologies, it remains unknown whether HGF/Met signaling is essential for myocardial development and/or physiological function in adulthood. We therefore investigated the requirement of HGF/Met signaling in cardiomyocyte for embryonic and postnatal heart development and function by conditional inactivation of the Met receptor in cardiomyocytes using the Cre-α-MHC mouse line (referred to as α-MHCMet-KO). Although α-MHCMet-KO mice showed normal heart development and were viable and fertile, by 6 months of age, males developed cardiomyocyte hypertrophy, associated with interstitial fibrosis. A significant upregulation in markers of myocardial damage, such as β-MHC and ANF, was also observed. By the age of 9 months, α-MHCMet-KO males displayed systolic cardiac dysfunction. Mechanistically, we provide evidence of a severe imbalance in the antioxidant defenses in α-MHCMet-KO hearts involving a reduced expression and activity of catalase and superoxide dismutase, with consequent reactive oxygen species accumulation. Similar anomalies were observed in females, although with a slower kinetics. We also found that Met signaling down-regulation leads to an increase in TGF-β production and a decrease in p38MAPK activation, which may contribute to phenotypic alterations displayed in α-MHCMet-KO mice. Consistently, we show that HGF acts through p38α to upregulate antioxidant enzymes in cardiomyocytes. Our results highlight that HGF/Met signaling in cardiomyocytes plays a physiological cardio-protective role in adult mice by acting as an endogenous regulator of heart function through oxidative stress control.

C3G silencing enhances STI-571-induced apoptosis in CML cells through p38 MAPK activation, but it antagonizes STI-571 inhibitory effect on survival.

In this work we report evidences of a functional relationship between C3G and p38 MAPK in the apoptotic effect of STI-571 on the chronic myeloid leukemia (CML) cell line K562. This has been demonstrated by knocking down C3G and p38alpha using the interfering RNA approach, as well as through targeting p38 by its inhibitor SB203580. The results indicate that p38 is a mediator of the STI-571-induced apoptosis, while C3G plays a negative role on STI-571-mediated p38 activation through a Rap1-dependent mechanism. According to this, gene expression analysis in C3G silenced cells revealed an upregulation of a large number of genes involved in apoptosis. Some of these genes are also down-regulated (at the protein level) upon p38alpha knock-down, which further suggests a functional association between these two proteins. On the other hand, C3G knock-down reverts the STI-571-inhibitory effect on ERKs and Akt pathways in a Rap1-independent fashion. Moreover, C3G overexpression also increased both, basal and STI-571-induced apoptosis, in agreement with previous reports. Therefore, our results strongly suggest a dual regulatory role for C3G in CML cells, modulating both apoptosis and survival via Rap-dependent and independent mechanisms.

p38 MAPK down-regulates fibulin 3 expression through methylation of gene regulatory sequences. Role in migration and invasion

Background: p38α MAPK regulates migration/invasion. Results: p38α induces hypermethylation of Fibulin 3 gene regulatory sequences leading to Fibulin 3 down-regulation. This contributes to regulate migration and invasion in MEFs and HCT116 cells. Conclusion: p38α down-regulates fibulin 3 expression through promoter methylation to control p38α-mediated migration and invasion. Significance: To understand the function of new p38α targets in migration/invasion and tumorigenesis. p38 MAPKs regulate migration and invasion. However, the mechanisms involved are only partially known. We had previously identified fibulin 3, which plays a role in migration, invasion, and tumorigenesis, as a gene regulated by p38α. We have characterized in detail how p38 MAPK regulates fibulin 3 expression and its role. We describe here for the first time that p38α, p38γ, and p38δ down-regulate fibulin 3 expression. p38α has a stronger effect, and it does so through hypermethylation of CpG sites in the regulatory sequences of the gene. This would be mediated by the DNA methylase, DNMT3A, which is down-regulated in cells lacking p38α, but once re-introduced represses Fibulin 3 expression. p38α through HuR stabilizes dnmt3a mRNA leading to an increase in DNMT3A protein levels. Moreover, by knocking-down fibulin 3, we have found that Fibulin 3 inhibits migration and invasion in MEFs by mechanisms involving p38α/β inhibition. Hence, p38α pro-migratory/invasive effect might be, at least in part, mediated by fibulin 3 down-regulation in MEFs. In contrast, in HCT116 cells, Fibulin 3 promotes migration and invasion through a mechanism dependent on p38α and/or p38β activation. Furthermore, Fibulin 3 promotes in vitro and in vivo tumor growth of HCT116 cells through a mechanism dependent on p38α, which surprisingly acts as a potent inducer of tumor growth. At the same time, p38α limits fibulin 3 expression, which might represent a negative feed-back loop.