No abstract

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor β Receptor (TGFβR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFβR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFβR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially-derived cell invasion in Tgfbr3−/− hearts. Tgfbr3−/− epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFβ1 and TGFβ2. Unexpectedly, loss of TGFβR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFβR3 in Tgfbr3−/− cells rescued deficits in invasion in vitro in response TGFβ1 and TGFβ2 as well as FGF2 and HMW-HA. Expression of TGFβR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3−/− cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3+/+ cells decreased invasion in response to TGFβ2, FGF2, and HMW-HA. We conclude that TGFβR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3−/− mice.

In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFβ2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFβ2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFβ2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFβ2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFβ2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFβ2. Taken together, these findings demonstrate that TGFβ2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFβ2 and HA signals are integrated to regulate changes in epicardial cell behavior.

During embryonic development, cells comprising the outermost layer of the heart or epicardium play a critical role in the formation of the coronary vasculature. Thus, uncovering the molecular mechanisms that govern epicardial cell behavior is imperative to better understand the etiology of cardiovascular diseases. In this study, we investigated the function of hyaluronan (HA), a major component of the extracellular matrix, in the modulation of epicardial signaling. We show that stimulation of epicardial cells with high molecular weight HA (HMW-HA) promotes the association of MEKK1 with the HA receptor CD44 and induces MEKK1 phosphorylation. This leads to the activation of two distinct pathways, one ERK-dependent and another NFκB-dependent. Furthermore, HMW-HA stimulates epicardial cells to differentiate and invade, as suggested by increased vimentin expression and enhanced invasion through a collagen matrix. Blockade of CD44, transfection with a kinase inactive MEKK1 construct or the use of ERK1/2 and NFκB inhibitors significantly abrogates the invasive response to HMW-HA. Together, these findings suggest an important role for HA in the regulation of epicardial cell fate via activation of MEKK1 signaling cascades.

Hyaluronan (HA) induces changes in cellular behavior that are crucial during both embryonic development and cancer progression. However, the biological effects of varying sizes of HA and the signal transduction mechanisms that these polymers may activate remain unclear. In this study, we demonstrate that pulse stimulation of mouse embryonic fibroblasts with high-molecular-weight (HMW) HA, but not HA of lower molecular sizes, leads to increases in Snail2 protein which are dependent on NFkappaB activity. Involvement of CD44, the main HA receptor, in these responses was determined by use of a CD44 blocking antibody and CD44 siRNA. Both the blockade and silencing of CD44 significantly abrogate the increases in nuclear factor kappaB (NFkappaB) activity and Snail2 protein following HMW-HA stimulation. Furthermore, we show that HMW-HA induces cellular invasion and that inhibition of CD44, Snail2, or NFkappaB significantly decreases this response. These studies elucidate a novel HA/Snail2 functional connection through CD44 and NFkappaB that is important for the induction of cellular invasion and is dependent on HA size.

Summary We previously reported that malignant melanomas express high levels of the mRNA binding protein CRD-BP. This molecule is important for the activation of anti-apoptotic pathways, a mechanism often linked to insensitivity to therapeutics. However, it is not known whether CRD-BP plays a role in the resistance of melanomas to anti-cancer treatment. Here we demonstrate that knock-down of CRD-BP with a specific sh-RNA enhances the effect of dacarbazine, temozolomide, vinblastine and etoposide on both primary and metastatic melanoma cell lines. CRD-BP down-regulation contributes to cell sensitization by increasing apoptosis and diminishing melanoma cell growth in response to chemotherapeutic agents. Furthermore, inhibition of CRD-BP decreases MITF expression and reintroduction of MITF partially compensates for the absence of CRD-BP. These findings suggest that high expression of CRD-BP in melanoma cells confers resistance to chemotherapy and that these CRD-BP responses are mediated, at least in part, by MITF.

SummaryWe have previously shown that the mRNA binding protein CRD-BP is overexpressed in human melanomas, where it promotes cell survival and resistance to chemotherapy. The present study investigates the role of hypoxia, a common characteristic of the tumor microenvironment, in the regulation of CRD-BP expression and melanoma cell responses. We found that hypoxia increases CRD-BP levels in metastatic melanoma cell lines but not in melanocytes or primary melanoma cells. Hypoxic stimulation transcriptionally regulates CRD-BP by facilitating the acetylation of histones within the CRD-BP gene and by modulating the extent of HIF1a binding to the CRD-BP promoter. Hypoxia significantly enhances the proliferative and invasive potential of metastatic melanoma cells but not that of normal or primary melanoma cells. Furthermore, inhibition of CRD-BP impairs the ability of metastatic cells to proliferate and invade in response to hypoxia. These findings identify CRD-BP as a novel effector of hypoxic responses that is relevant for the selection of metastatic cells. This work also describes a previously unknown role for CRD-BP in the regulation of melanoma cell invasion and highlights the importance of the hypoxic microenvironment in determining cell fate.

The kidney is a major site of chemical excretion, which results in its propensity to exhibit chemically-induced toxicological effects at a higher rate than most other organs. Although the kidneys are often weighed in animal toxicity studies, the manner in which these kidney weight measurements are interpreted and the value of this information in predicting renal damage remains controversial. In this study we sought to determine whether a relationship exists between chemically-induced kidney weight changes and renal histopathological alterations. We also examined the relative utility of absolute and relative (kidney-to-body weight ratio) kidney weight in the prediction of renal toxicity. For this, data extracted from oral chemical exposure studies in rats performed by the National Toxicology Program were qualitatively and quantitatively evaluated. Our analysis showed a statistically significant correlation between absolute, but not relative, kidney weight and renal histopathology in chemically-treated rats. This positive correlation between absolute kidney weight and histopathology was observed even with compounds that statistically decreased terminal body weight. Also, changes in absolute kidney weight, which occurred at subchronic exposures, were able to predict the presence or absence of kidney histopathology at both subchronic and chronic exposures. Furthermore, most increases in absolute kidney weight reaching statistical significance (irrespective of the magnitude of change) were found to be relevant for the prediction of histopathological changes. Hence, our findings demonstrate that the evaluation of absolute kidney weight is a useful method for identifying potential renal toxicants.