DKK3 (Dickkopf 3) Alters Atherosclerotic Plaque Phenotype Involving Vascular Progenitor and Fibroblast Differentiation Into Smooth Muscle Cells.

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-ß (transforming growth factor-ß)/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.

p38α regulates cytokine-induced IFNγ secretion via the Mnk1/eIF4E pathway in Th1 cells.

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of immune and inflammatory processes. We used p38α-conditional, p38ß-deficient and p38α/ß 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 p38ß 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 p38ß modulate T-cell receptor-induced IFNγ and TNFα production, whereas only p38α regulates cytokine-induced IFNγ production. The lack of p38α and p38ß 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 p38ß in T-cell proliferation.

Liver-specific p38α deficiency causes reduced cell growth and cytokinesis failure during chronic biliary cirrhosis in mice.

UNLABELLED: p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor ß, tumor necrosis factor-α, interleukin-1ß, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice. CONCLUSION: Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.

Genetic analysis of specific and redundant roles for p38alpha and p38beta MAPKs during mouse development.

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 p38ß. We found that mice with combined deletion of p38α and p38ß 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 p38ß in the regulation of cardiac gene expression during development. By using knock-in animals that express p38ß under control of the endogenous p38α promoter, we also found that p38ß cannot perform all of the functions of p38α during embryogenesis. Our results identify essential roles for p38α and p38ß during development and suggest that some specific functions may be explained by differences in expression patterns.

Discovery and In Vivo Efficacy of AZ-PRMT5i-1, a Novel PRMT5 Inhibitor with High MTA Cooperativity.

PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.

Met acts through Abl to regulate p53 transcriptional outcomes and cell survival in the developing liver.

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.

Roles of p38 MAPKs in invasion and metastasis.

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.

AZD4625 is a Potent and Selective Inhibitor of KRASG12C.

AZD4625 is a potent, selective, and orally bioavailable inhibitor of oncogenic KRASG12C as demonstrated in cellular assays and in vivo in preclinical cell line-derived and patient-derived xenograft models. In vitro and cellular assays have shown selective binding and inhibition of the KRASG12C mutant isoform, which carries a glycine to cysteine mutation at residue 12, with no binding and inhibition of wild-type RAS or isoforms carrying non-KRASG12C mutations. The pharmacology of AZD4625 shows that it has the potential to provide therapeutic benefit to patients with KRASG12C mutant cancer as either a monotherapy treatment or in combination with other targeted drug agents.

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis.

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.

Regulation of Mammary Luminal Cell Fate and Tumorigenesis by p38α.

Mammary stem and progenitor cells are essential for mammary gland homeostasis and are also candidates for cells of origin of mammary tumors. Here, we have investigated the function of the protein kinase p38α in the mammary gland using mice that delete this protein in the luminal epithelial cells. We show that p38α regulates the fate of luminal progenitor cells through modulation of the transcription factor RUNX1, an important controller of the estrogen receptor-positive cell lineage. We also provide evidence that the regulation of RUNX1 by p38α probably involves the kinase MSK1, which phosphorylates histone H3 at the RUNX1 promoter. Moreover, using a mouse model for breast cancer initiated by luminal cells, we show that p38α downregulation in mammary epithelial cells reduces tumor burden, which correlates with decreased numbers of tumor-initiating cells. Collectively, our results define a key role for p38α in luminal progenitor cell fate that affects mammary tumor formation.

MSK1 regulates luminal cell differentiation and metastatic dormancy in ER+ breast cancer.

For many patients with breast cancer, symptomatic bone metastases appear after years of latency. How micrometastatic lesions remain dormant and undetectable before initiating colonization is unclear. Here, we describe a mechanism involved in bone metastatic latency of oestrogen receptor-positive (ER+) breast cancer. Using an in vivo genome-wide short hairpin RNA screening, we identified the kinase MSK1 as an important regulator of metastatic dormancy in breast cancer. In patients with ER+ breast cancer, low MSK1 expression associates with early metastasis. We show that MSK1 downregulation impairs the differentiation of breast cancer cells, increasing their bone homing and growth capacities. MSK1 controls the expression of genes required for luminal cell differentiation, including the GATA3 and FOXA1 transcription factors, by modulating their promoter chromatin status. Our results indicate that MSK1 prevents metastatic progression of ER+ breast cancer, suggesting that stratifying patients with breast cancer as high or low risk for early relapse based on MSK1 expression could improve prognosis.

Publisher Correction: MSK1 regulates luminal cell differentiation and metastatic dormancy in ER+ breast cancer.

In the version of this Article originally published, the boxes framing the two plots in Fig. 1g were misaligned from the axes due to a technical error. This has now been corrected in all versions of the Article.

Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope.

Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins.

Dual function of p38α MAPK in colon cancer: suppression of colitis-associated tumor initiation but requirement for cancer cell survival.

Colorectal cancer is frequently associated with chronic inflammation, with the intestinal epithelial barrier playing an important protective role against the infections and injuries that cause colitis. The p38α pathway regulates inflammatory responses but can also suppress tumor initiation in epithelial cells. We have found that p38α signaling has a dual function in colorectal tumorigenesis. On one side, p38α protects intestinal epithelial cells against colitis-associated colon cancer by regulating intestinal epithelial barrier function. Accordingly, p38α downregulation results in enhanced colitis-induced epithelial damage and inflammation, which potentiates colon tumor formation. Surprisingly, inhibition of p38α in transformed colon epithelial cells reduces tumor burden. Thus, p38α suppresses inflammation-associated epithelial damage and tumorigenesis but contributes to the proliferation and survival of tumor cells.

Gadd45γ and Map3k4 interactions regulate mouse testis determination via p38 MAPK-mediated control of Sry expression.

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 p38ß 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.

Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning.

A gradient of Wnt/beta-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopus embryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2 rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.

Dkk1 and noggin cooperate in mammalian head induction.

Growth factor antagonists play important roles in mediating the inductive effects of the Spemann organizer in amphibian embryos and its equivalents in other vertebrates. Dual inhibition of Wnt and BMP signals has been proposed to confer head organizer activity. We tested the requirement of this coinhibition in Xenopus and mice. In Xenopus, simultaneous reduction of the BMP antagonists chordin and noggin, and the Wnt antagonist dickkopf1 (dkk1) leads to anterior truncations. In mice, compound mutants for dkk1 and noggin display severe head defects, with deletion of all head structures anterior to the mid-hindbrain boundary. These defects arise as a result of a failure in anterior specification at the gastrula stage. The results provide genetic evidence for the dual inhibition model and indicate that dkk1 and noggin functionally cooperate in the head organizer.