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.

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.

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.

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.

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.