GCKIII (Germinal Center Kinase III) Kinases STK24 and STK25 (Serine/Threonine Kinase 24 and 25) Inhibit Cavernoma Development.

BACKGROUND: Cavernous cerebral malformations can arise because of mutations in the CCM1, CCM2, or CCM3 genes, and lack of Cdc42 has also been reported to induce these malformations in mice. However, the role of the CCM3 (cerebral cavernous malformation 3)-associated kinases in cavernoma development is not known, and we, therefore, have investigated their role in the process. METHODS: We used a combination of an in vivo approach, using mice genetically modified to be deficient in the CCM3-associated kinases STK24 and STK25 (serine/threonine kinases 24 and 25), and the in vitro model of human endothelial cells in which expression of STK24 and STK25 was inhibited by RNA interference. RESULTS: Mice deficient for both Stk24 and Stk25, but not for either of them individually, developed aggressive vascular lesions with the characteristics of cavernomas at an early age. Stk25 deficiency also gave rise to vascular anomalies in the context of Stk24 heterozygosity. Human endothelial cells deficient for both kinases phenocopied several of the consequences of CCM3 loss, and single STK25 deficiency also induced KLF2 expression, Golgi dispersion, altered distribution of ß-catenin, and appearance of stress fibers. CONCLUSIONS: The CCM3-associated kinases STK24 and STK25 play a major role in the inhibition of cavernoma development.

High Levels of Receptor Tyrosine Kinases in CCM3-Deficient Cells Increase Their Susceptibility to Tyrosine Kinase Inhibition.

Cerebral cavernous malformations (CCMs) are vascular malformations that can be the result of the deficiency of one of the CCM genes. Their only present treatment is surgical removal, which is not always possible, and an alternative pharmacological strategy to eliminate them is actively sought. We have studied the effect of the lack of one of the CCM genes, CCM3, in endothelial and non-endothelial cells. By comparing protein expression in control and CCM3-silenced cells, we found that the levels of the Epidermal Growth Factor Receptor (EGFR) are higher in CCM3-deficient cells, which adds to the known upregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in these cells. Whereas VEGFR2 is upregulated at the mRNA level, EGFR has a prolonged half-life. Inhibition of EGFR family members in CCM3-deficient cells does not revert the known cellular effects of lack of CCM genes, but it induces significantly more apoptosis in CCM3-deficient cells than in control cells. We propose that the susceptibility to tyrosine kinase inhibitors of CCM3-deficient cells can be harnessed to kill the abnormal cells of these lesions and thus treat CCMs pharmacologically.

Measuring the Kinase Activity of GCKIII Proteins In Vitro.

One of the CCM genes, CCM3/PDCD10, binds to the protein kinase family GCKIII, which comprises MST3/STK24, SOK1/STK25, and MST4/STK26. These proteins have been shown to have the same effect as CCM3, both in endothelial cells and in animal models such as zebrafish and are most likely involved in CCM pathogenesis. We describe here an in vitro kinase assay of GCKIII proteins which can be used to study their regulation in endothelial and other cells under different circumstances.

MST Kinases and Metabolism.

Since the discovery of the mammalian sterile twenty (MST) kinase family of proteins (MST1/STK4, MST2/STK3, MST3/STK24, and SOK1/STK25), much has been done that adds to our knowledge of their structure, regulation, and function. In the last few years, a series of articles has unveiled a previous unknown relation of these kinases with metabolic regulation and the homeostasis of metabolic tissues. The aim of this review is to bring together this body of data to provide a detailed picture of the current knowledge about these proteins, metabolism, and some of the associated diseases.

The MST3/STK24 kinase mediates impaired fasting blood glucose after a high-fat diet.

AIMS/HYPOTHESIS: The identification of mediators in the pathogenesis of type 2 diabetes mellitus is essential for the full understanding of this disease. Protein kinases are especially important because of their potential as pharmacological targets. The goal of this study was to investigate whether mammalian sterile-20 3 (MST3/STK24), a stress-regulated kinase, is involved in metabolic alterations in obesity. METHODS: Glucose regulation of Mst3 (also known as Stk24)-knockout mice was analysed both in 129;C57 mixed background mice and in C57/BL6J mice fed normally or with a high-fat diet (HFD). This work was complemented with an analysis of the insulin signalling pathway in cultured human liver cells made deficient in MST3 using RNA interference. RESULTS: MST3 is phosphorylated in the livers of mice subject to an obesity-promoting HFD, and its deficiency lowers the hyperglycaemia, hyperinsulinaemia and insulin resistance that the animals develop with this diet, an effect that is seen even without complete inactivation of the kinase. Lack of MST3 results in activation of the insulin signalling pathway downstream of IRS1, in both cultured liver cells and the liver of animals after HFD. This effect increases the inhibition of forkhead box (FOX)O1, with subsequent downregulation of the expression of gluconeogenic enzymes. CONCLUSIONS/INTERPRETATION: MST3 inhibits the insulin signalling pathway and is important in the development of insulin resistance and impaired blood glucose levels after an HFD.

Corrigendum: Hepatic p63 regulates steatosis via IKKß/ER stress.

This corrects the article DOI: 10.1038/ncomms15111.

Hepatic p63 regulates steatosis via IKKß/ER stress.

p53 family members control several metabolic and cellular functions. The p53 ortholog p63 modulates cellular adaptations to stress and has a major role in cell maintenance and proliferation. Here we show that p63 regulates hepatic lipid metabolism. Mice with liver-specific p53 deletion develop steatosis and show increased levels of p63. Down-regulation of p63 attenuates liver steatosis in p53 knockout mice and in diet-induced obese mice, whereas the activation of p63 induces lipid accumulation. Hepatic overexpression of N-terminal transactivation domain TAp63 induces liver steatosis through IKKß activation and the induction of ER stress, the inhibition of which rescues the liver functions. Expression of TAp63, IKKß and XBP1s is also increased in livers of obese patients with NAFLD. In cultured human hepatocytes, TAp63 inhibition protects against oleic acid-induced lipid accumulation, whereas TAp63 overexpression promotes lipid storage, an effect reversible by IKKß silencing. Our findings indicate an unexpected role of the p63/IKKß/ER stress pathway in lipid metabolism and liver disease.

A Functional Link between AMPK and Orexin Mediates the Effect of BMP8B on Energy Balance.

AMP-activated protein kinase (AMPK) in the ventromedial nucleus of the hypothalamus (VMH) and orexin (OX) in the lateral hypothalamic area (LHA) modulate brown adipose tissue (BAT) thermogenesis. However, whether these two molecular mechanisms act jointly or independently is unclear. Here, we show that the thermogenic effect of bone morphogenetic protein 8B (BMP8B) is mediated by the inhibition of AMPK in the VMH and the subsequent increase in OX signaling via the OX receptor 1 (OX1R). Accordingly, the thermogenic effect of BMP8B is totally absent in ox-null mice. BMP8B also induces browning of white adipose tissue (WAT), its thermogenic effect is sexually dimorphic (only observed in females), and its impact on OX expression and thermogenesis is abolished by the knockdown of glutamate vesicular transporter 2 (VGLUT2), implicating glutamatergic signaling. Overall, our data uncover a central network controlling energy homeostasis that may be of considerable relevance for obesity and metabolic disorders.

The cerebral cavernous malformation 3 gene is necessary for senescence induction.

Mutations in cerebral cavernous malformation 3 gene are known to result in development of vascular malformations and have recently been proposed to also give rise to meningiomas. We report in this study that lack of CCM3 unexpectedly impairs the senescence response of cells, and this is related to the inability of CCM3-deficient cells to induce the C/EBPß transcription factor and implement the senescence-associated secretory phenotype. Induction of C/EBPß and cytokines is also impaired in the absence of CCM3 in response to cytokines in nonsenescent cells, pointing to it being a primary defect and not secondary to impaired senescence. CCM3-deficient cells also have a defect in autophagy at late passages of culture, and this defect is also not dependent on impaired senescence, as it is evident in immortal cells after nutrient starvation. Further, these two defects may be related, as enforcing autophagy in CCM3-deficient late passage cells increases C/EBPß cytokine expression. These results broaden our knowledge on the mechanisms by which CCM3 deficiency results in disease and open new avenues of research into both CCM3 and senescence biology.

The CCM3-GCKIII partnership.

Specific mutations in the CCM3 gene predispose to the development of cerebral cavernous malformations, a special type of vascular lesions. This calls for an elucidation of the precise nature of the CCM3 protein and a deep understanding of its molecular regulation. In this review, we outline our current knowledge of the different CCM3 protein complexes. We focus on the GCKIII family of kinases as partners of CCM3 and discuss the functional consequences of this partnership, putting forward a putative model for the activation of these kinases.

Cerebral cavernous malformations: from CCM genes to endothelial cell homeostasis.

Cerebral cavernous malformations (CCMs) are vascular lesions that can occur sporadically or as a consequence of inherited loss-of-function mutations, predominantly in the genes CCM1 (KRIT1), CCM2 (MGC4607, OSM, Malcavernin), or CCM3 (PDCD10, TFAR15). Inherited, familial CCM is characterized by the development of multiple lesions throughout a patient's life leading to recurrent cerebral hemorrhages. Recently, roles for the CCM proteins in maintaining vascular barrier functions and quiescence have been elucidated, and in this review we summarize the genetics and pathophysiology of this disease and discuss the molecular mechanisms through which CCM proteins may act within blood vessels.

Adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress.

While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.

A novel cardioprotective p38-MAPK/mTOR pathway.

Despite intensive study, the mechanisms regulating activation of mTOR and the consequences of that activation in the ischemic heart remain unclear. This is particularly true for the setting of ischemia/reperfusion (I/R) injury. In a mouse model of I/R injury, we observed robust mTOR activation, and its inhibition by rapamycin increased injury. Consistent with the in-vivo findings, mTOR activation was also protective in isolated cardiomyocytes exposed to two models of I/R. Moreover, we identify a novel oxidant stress-activated pathway regulating mTOR that is critically dependent on p38-MAPK and Akt. This novel p38-regulated pathway signals downstream through REDD1, Tsc2, and 14-3-3 proteins to activate mTOR and is independent of AMPK. The protective role of p38/Akt and mTOR following oxidant stress is a general phenomenon since we observed it in a wide variety of cell types. Thus we have identified a novel protective pathway in the cardiomyocyte involving p38-mediated mTOR activation. Furthermore, the p38-dependent protective pathway might be able to be selectively modulated to enhance cardio-protection while not interfering with the inhibition of the better-known detrimental p38-dependent pathways.

CCM3/PDCD10 stabilizes GCKIII proteins to promote Golgi assembly and cell orientation.

Mutations in CCM3/PDCD10 result in cerebral cavernous malformations (CCMs), a major cause of cerebral hemorrhage. Despite intense interest in CCMs, very little is known about the function of CCM3. Here, we report that CCM3 is located on the Golgi apparatus, forming a complex with proteins of the germinal center kinase III (GCKIII) family and GM130, a Golgi-resident protein. Cells depleted of CCM3 show a disassembled Golgi apparatus. Furthermore, in wound-healing assays, CCM3-depleted cells cannot reorient the Golgi and centrosome properly, and demonstrate impaired migration. Golgi disassembly after either depletion of CCM3 or dissociation of CCM3 from the GM130-GCKIII complex is the result of destabilization of GCKIII proteins and dephosphorylation of their substrate, 14-3-3zeta. Significantly, the phenotype induced by CCM3 depletion can be reverted by expression of wild-type CCM3, but not by disease-associated mutants. Our findings suggest that Golgi dysfunction and the ensuing abnormalities of cell orientation and migration resulting from CCM3 mutations contribute to CCM pathogenesis.

CDK redundancy guarantees cell cycle progression in Rb-negative tumor cells independently of their p16 status.

A high percentage of tumor cells bear mutations in the Rb tumor suppressor gene. They have high levels of the cdk inhibitor p16(ink4a) and no cyclin D/cdk4-6 complexes. Although p16 is known not to arrest the proliferation of Rb-negative cells, it is not known whether its presence affects how their cycle progresses, how E2F-dependent transcription is modulated, and whether and how Rb-related proteins are inactivated. We have assessed the relevance of p16(ink4a) for cell cycle progression of these cells. Using SaOS2 osteosarcoma cells as a model, we find that downregulation of p16(ink4a) by RNAi and reconstitution of active cyclin D/cdk4 complexes does not affect progression of the cycle of these cells or expression of E2F-dependent genes. Rb-negative tumor cells can functionally inactivate Rb-related proteins in G(1)/S transition independently of their p16(ink4a) status. Furthermore, Rb-negative tumor cells do not arrest when cdk1, cdk2 or cdk3 are inhibited by RNAi, independently of their p16(ink4a) status, and combined inhibition of these cdks is also not enough to arrest their cell cycle. However, cell cycle progression of Rb-negative tumor cells is sensitive to complete cdk inhibition, as it is arrested by the chemical cdk inhibitor roscovitine and the biological cdk inhibitor p27. These results suggest that, despite their lack of cyclin D-containing complexes, Rb-negative tumor cells, like normal untransformed cells, take advantage of the high degree of redundancy of cdks for their cell cycle progression.

SOK1 translocates from the Golgi to the nucleus upon chemical anoxia and induces apoptotic cell death.

SOK1 is a Ste20 protein kinase of the germinal center kinase (GCK) family that has been shown to be activated by oxidant stress and chemical anoxia, a cell culture model of ischemia. More recently, it has been shown to be localized to the Golgi apparatus, where it functions in a signaling pathway required for cell migration and polarization. Herein, we demonstrate that SOK1 regulates cell death after chemical anoxia, as its down-regulation by RNA interference enhances cell survival. Furthermore, expression of SOK1 elicits apoptotic cell death by activating the intrinsic pathway. We also find that a cleaved form of SOK1 translocates from the Golgi to the nucleus after chemical anoxia and that this translocation is dependent on both caspase activity and on amino acids 275-292, located immediately C-terminal to the SOK1 kinase domain. Furthermore, SOK1 entry into the nucleus is important for the cell death response since SOK1 mutants unable to enter the nucleus do not induce cell death. In summary, SOK1 is necessary to induce cell death and can induce death when overexpressed. Furthermore, SOK1 appears to play distinctly different roles in stressed versus non-stressed cells, regulating cell death in the former.

Of mice without pockets: mouse models to study the function of Rb family proteins.

Three negative regulators of cell cycle, the related proteins, pRB, p107 and p130, constitute the family of pocket proteins. pRB is a tumor suppressor which has drawn a lot of attention on its family of proteins, with the ensuing intense study of their biology. As a result we have a wealth of information on their biochemistry and biology, ranging from their regulation to their biochemical activities, and the effects of their absence or overexpression on cells. Despite this, many questions remain unsolved. In recent years, analysis of genetically-modified mouse strains has provided interesting data regarding the physiological and pathophysiological roles of these three proteins. Specifically, germ-line and conditional knockout strains for one or more than one of the members of the family have revealed as powerful tools in this regard. Here we review the mouse models available for studying these cell cycle regulators and how data generated by these approaches have sometimes challenged previous thoughts about the pocket proteins biology.

The GCK II and III subfamilies of the STE20 group kinases.

The Ste20 (sterile 20) proteins are a large family of serine/threonine kinases. Since their discovery a growing body of evidence has implicated them in the regulation of signaling pathways governing cell growth, cell differentiation cell death and cell volume. Approximately 30 human members have been identified based on the high degree of homology of their catalytic domain to that of the Ste20p from Saccharomyces cerevisiae. In addition to the conserved regions, there are also regions of sequence that make each of them unique. In this review we will focus on two subfamilies of the group, GCK-II and GCK-III, families that are closely related but, again, unique in their structural features and biological functions. Herein, we will present what we hope will be the current state of knowledge about these kinases, and discuss what remains to be done in order to better understand their activity and regulation.

Plitidepsin has a cytostatic effect in human undifferentiated (anaplastic) thyroid carcinoma.

Undifferentiated (anaplastic) thyroid carcinoma is a highly aggressive human cancer with very poor prognosis. Although there have been a few studies of candidate treatments, the fact that it is an infrequent tumor makes it very difficult to design clinical trials. A strong association has been observed between undifferentiated thyroid carcinoma and TP53 mutations in numerous molecular genetic and expression studies. Plitidepsin (Aplidin, PharmaMar, Madrid, Spain) is a novel anticancer compound obtained from a sea tunicate. This compound has been reported to induce apoptosis independently of TP53 status. We investigated the actions of plitidepsin in human thyroid cancer cells. In initial experiments using primary cultured cells from a differentiated (papillary) carcinoma, we found that 100 nmol/L plitidepsin induced apoptosis, whereas lower doses were cytostatic. Because our aim was to study the effects of plitidepsin at clinically relevant concentrations, subsequent experiments were done with a dosage regimen reflecting plasma concentrations observed in previously reported clinical trials: 100 nmol/L for 4 hours, followed by 10 nmol/L for 20 hours (4(100)/20(10) plitidepsin). This plitidepsin dosage regimen blocked the proliferation of a primary undifferentiated/anaplastic thyroid carcinoma culture obtained in our laboratory and of a commercial cell line (8305C) obtained from an undifferentiated thyroid carcinoma; however, it did not induce apoptosis. The proportion of cells in the G(1) phase of the cell cycle was greatly increased and the proportion in the S/G(2)-M phases greatly reduced, suggesting that plitidepsin blocks G(1)-to-S transition. Levels of the cyclin D1/cyclin-dependent kinase 4/p21 complex proteins were decreased and, in line with this, the levels of unphosphorylated Rb1 increased. The decrease in cell cycle proteins correlated with hypoacetylation of histone H3. Finally, we did experiments to assess how rapidly tumor cells return to their initial pretreatment proliferative behavior after 4(100)/20(10) plitidepsin treatment. Cells from undifferentiated tumors needed more than 3 days to recover logarithmic growth, and after 7 days, cell number was still significantly lower than in control cultures. 4(100)/20(10) plitidepsin inhibited the growth in soft agar. Together, our data show that plitidepsin is able to block in vitro cell cycle progression at concentrations similar to serum concentrations observed in vivo, and that this effect is persistent for several days after plitidepsin removal. Whether plitidepsin will prove to be clinically useful in the treatment of undifferentiated thyroid cancers remains to be established. However, our results raise the possibility that plitidepsin might be effective alone or in combination with radiotherapy and/or other drug treatments.

Inhibition of Cdk4 activity enhances translation of p27kip1 in quiescent Rb-negative cells.

We show in this work that the inhibition of Cdk4 (6) in Rb(-/-) 3T3 cells enhances the accumulation of the p27(kip1) cyclin-dependent kinase inhibitor when these cells are induced into quiescence. Two different forms of inhibition of Cdk4 (6), namely overexpression of the Cdk4 (6) inhibitor p16 and overexpression of a dominant negative mutant of Cdk4 (Cdk4(N158)), result in this effect. This suggests that the relevant activity of Cdk4 (6) that has to be inactivated in this setting is its kinase activity. The accumulation of p27(kip1) is due to enhanced translation of the protein, mediated by the 3'-untranslated region of the p27(kip1) mRNA. Moreover, the cells that overexpress p16(ink4a) or Cdk4(N158) show a delay in G(1) when made quiescent and restimulated to proliferate. This delay is overcome by transfection of a plasmid expressing antisense p27(kip1), which shows that the accumulation of p27(kip1) in these cells is related to their G(1) delay. In summary, we report a new functional link between two important cell cycle regulators, Cdk4 and p27(kip1), and provide a mechanistic explanation to the previously reported epistatic relations between these two proteins.