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Objective— DKK3 (dickkopf 3), a 36-kD secreted glycoprotein, has been shown to be involved in the differentiation of partially reprogrammed cells and embryonic stem cells to smooth muscle cells (SMCs), but little is known about its involvement in vascular disease. This study aims to assess the effects of DKK3 on atherosclerotic plaque composition. Approach and Results— In the present study, we used a murine model of atherosclerosis ( ApoE −/− ) in conjunction with DKK3 −/− and performed tandem stenosis of the carotid artery to evaluate atherosclerotic plaque development. We found that the absence of DKK3 leads to vulnerable atherosclerotic plaques, because of a reduced number of SMCs and reduced matrix protein deposition, as well as increased hemorrhage and macrophage infiltration. Further in vitro studies revealed that DKK3 can induce differentiation of Sca1 + (stem cells antigen 1) vascular progenitors and fibroblasts into SMCs via activation of the TGF-β (transforming growth factor-β)/ATF6 (activating transcription factor 6) and Wnt signaling pathways. Finally, we assessed the therapeutic potential of DKK3 in mouse and rabbit models and found that DKK3 altered the atherosclerotic plaque content via increasing SMC numbers and reducing vascular inflammation. Conclusions— Cumulatively, we provide the first evidence that DKK3 is a potent SMC differentiation factor, which might have a therapeutic effect in reducing intraplaque hemorrhage related to atherosclerotic plaque phenotype.

Supplemental Digital Content is available in the text., Background: Dickkopf-related protein 3 (DKK3) is a secreted protein that is involved in the regulation of cardiac remodeling and vascular smooth muscle cell differentiation, but little is known about its role in atherosclerosis. Methods: We tested the hypothesis that DKK3 is atheroprotective using both epidemiological and experimental approaches. Blood DKK3 levels were measured in the Bruneck Study in 2000 (n=684) and then in 2005 (n=574). DKK3-deficient mice were crossed with apolipoprotein E-/- mice to evaluate atherosclerosis development and vessel injury-induced neointimal formation. Endothelial cell migration and the underlying mechanisms were studied using in vitro cell culture models. Results: In the prospective population-based Bruneck Study, the level of plasma DKK3 was inversely related to carotid artery intima-media thickness and 5-year progression of carotid atherosclerosis independently from standard risk factors for atherosclerosis. Experimentally, we analyzed the area of atherosclerotic lesions, femoral artery injury-induced reendothelialization, and neointima formation in both DKK3-/-/apolipoprotein E-/- and DKK3+/+/apolipoprotein E-/- mice. It was demonstrated that DKK3 deficiency accelerated atherosclerosis and delayed reendothelialization with consequently exacerbated neointima formation. To explore the underlying mechanisms, we performed transwell and scratch migration assays using cultured human endothelial cells, which exhibited a significant induction in cell migration in response to DKK3 stimulation. This DKK3-induced migration activated ROR2 and DVL1, activated Rac1 GTPases, and upregulated JNK and c-jun phosphorylation in endothelial cells. Knockdown of the ROR2 receptor using specific siRNA or transfection of a dominant-negative form of Rac1 in endothelial cells markedly inhibited cell migration and downstream JNK and c-jun phosphorylation. Conclusions: This study provides the evidence for a role of DKK3 in the protection against atherosclerosis involving endothelial migration and repair, with great therapeutic potential implications against atherosclerosis.

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of immune and inflammatory processes. We used p38α-conditional, p38β-deficient and p38α/β double-null mouse models to address the role of these two p38 MAPK in CD4+ T cells, and found that p38α deficiency causes these cells to hyperproliferate. Our studies indicate that both p38α and p38β are dispensable for T helper cell type 1 (Th1) differentiation but, by controlling interferon (IFN)γ and tumor necrosis factor (TNF)α production, are critical for normal Th1 effector function. We found that both p38α and p38β modulate T-cell receptor-induced IFNγ and TNFα production, whereas only p38α regulates cytokine-induced IFNγ production. The lack of p38α and p38β did not affect transcription and mRNA stability of Ifng. However, the absence of p38α in Th1 cells resulted in a decreased MNK1 phosphorylation after cytokine activation, and MNK1 inhibition blocked IFNγ production. Our results indicate that p38α regulates IFNγ secretion through the activation of the MNK1/eIF4E pathway of translation initiation and identify specific functions for p38α and p38β in T-cell proliferation.

Cells from primary tumours need to go through several steps to become fully metastatic. During this process, cancer cells acquire the ability to invade, migrate across the surrounding tissue, enter into the circulation and colonize distant organs. In the present paper, we review recent progress in understanding how the p38 MAPK (mitogen-activated protein kinase) signalling pathway participates in the different steps of metastasis. Experimental evidence suggests that tumour cells need to modulate p38 MAPK activity levels to successfully metastasize.

Summary Loss of the kinase MAP3K4 causes mouse embryonic gonadal sex reversal due to reduced expression of the testis-determining gene, Sry. However, because of widespread expression of MAP3K4, the cellular basis of this misregulation was unclear. Here, we show that mice lacking Gadd45γ also exhibit XY gonadal sex reversal caused by disruption to Sry expression. Gadd45γ is expressed in a dynamic fashion in somatic cells of the developing gonads from 10.5 days postcoitum (dpc) to 12.5 dpc. Gadd45γ and Map3k4 genetically interact during sex determination, and transgenic overexpression of Map3k4 rescues gonadal defects in Gadd45γ-deficient embryos. Sex reversal in both mutants is associated with reduced phosphorylation of p38 MAPK and GATA4. In addition, embryos lacking both p38α and p38β also exhibit XY gonadal sex reversal. Taken together, our data suggest a requirement for GADD45γ in promoting MAP3K4-mediated activation of p38 MAPK signaling in embryonic gonadal somatic cells for testis determination in the mouse., Graphical Abstract Highlights ► Loss of Gadd45γ disrupts Sry expression and causes XY gonadal sex reversal ► Gadd45γ interacts with Map3k4 during testis determination ► Loss of Gadd45γ and Map3k4 disrupts p38 MAPK signaling and GATA4 phosphorylation ► Double-knockout embryos lacking p38α and p38β exhibit XY gonadal sex reversal, Testis determination in mammals is initiated by Sry expression. Warr et al. report XY gonadal sex reversal in mice lacking GADD45γ, a protein associated with cellular stress. Genetic and biochemical studies suggest a molecular pathway, linking GADD45γ, MAP3K4, p38 MAPK, and GATA4, required for the proper regulation of Sry expression.

p38α MAPK is an important regulator of cellular responses induced by external cues, but the elucidation of physiological functions for p38α has been complicated by the possible functional redundancy in vivo with the related family member p38β. We found that mice with combined deletion of p38α and p38β display diverse developmental defects at midgestation, including major cardiovascular abnormalities, which are observed neither in single knockout nor in double heterozygous embryos. Expression analysis indicates specific functions of p38α and p38β in the regulation of cardiac gene expression during development. By using knock-in animals that express p38β under control of the endogenous p38α promoter, we also found that p38β cannot perform all of the functions of p38α during embryogenesis. Our results identify essential roles for p38α and p38β during development and suggest that some specific functions may be explained by differences in expression patterns.

Background & Aims: Genetic studies indicate that distinct signaling modulators are each necessary but not individually sufficient for embryonic hepatocyte survival in vivo. Nevertheless, how signaling players are interconnected into functional circuits and how they coordinate the balance of cell survival and death in developing livers are still major unresolved issues. In the present study, we examined the modulation of the p53 pathway by HGF/ Met in embryonic livers. Methods: We combined pharmacological and genetic approaches to biochemically and functionally evaluate p53 pathway modulation in primary embryonic hepatocytes and in developing livers. RT-PCR arrays were applied to investigate the selectivity of p53 transcriptional response triggered by Met. Results: Met recruits p53 to regulate the liver developmental program, by qualitatively modulating its transcriptional properties: turning on the Mdm2 survival gene, while keeping death and cell-cycle arrest genes Pmaip1 and p21 silent. We investigated the mechanism leading to p53 regulation by Met and found that Abl and p38MAPK are required for p53 phosphorylation on S 389 , Mdm2 upregulation, and hepatocyte survival. Alteration of this signaling mechanism switches p53 properties, leading to p53-dependent cell death in embryonic livers. RT-PCR array studies affirmed the ability of the Met-Abl-p53 axis to modulate the expression of distinct genes that can be regulated by p53. Conclusions: A signaling circuit involving Abl and p38MAPK is required downstream of Met for the survival of embryonic hepatocytes, via qualitative regulation of the p53 transcriptional response, by switching its proapoptotic into survival properties. 2012 European Association for the Study of the Liver. Published

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.