Big mitogen-activated protein kinase 1 (BMK1), also known as ERK5, is a member of the MAPK family. Genetic ablation of BMK1 in mice leads to embryonic lethality, precluding the exploration of pathophysiological roles of BMK1 in adult mice. We generated a BMK1 conditional mutation in mice in which disruption of the BMK1 gene is under the control of the inducible Mx1-Cre transgene. Ablation of BMK1 in adult mice led to lethality within 2-4 weeks after the induction of Cre recombinase. Physiological analysis showed that the blood vessels became abnormally leaky after deletion of the BMK1 gene. Histological analysis revealed that, after BMK1 ablation, hemorrhages occurred in multiple organs in which endothelial cells lining the blood vessels became round, irregularly aligned, and, eventually, apoptotic. In vitro removal of BMK1 protein also led to the death of endothelial cells partially due to the deregulation of transcriptional factor MEF2C, which is a direct substrate of BMK1. Additionally, endothelial-specific BMK1-KO leads to cardiovascular defects identical to that of global BMK1-KO mutants, whereas, surprisingly, mice lacking BMK1 in cardiomyocytes developed to term without any apparent defects. Taken together, the data provide direct genetic evidence that the BMK1 pathway is critical for endothelial function and for maintaining blood vessel integrity. 1138The Nonstandard abbreviations used: big mitogen-activated protein kinase-1 (BMK1); BMK1 flox/flox Mx1-Cre mice treated with pIpC (BMK1-CKO); cardiomyocyte-specific BMK1-KO (BMK1-cmKO); EC-specific BMK1-KO (BMK1-ecKO); embryonic day (E); endothelial cell (EC); endothelial cell growth supplement (ECGS); extracellular signalregulated kinases-1 and -2 (ERK1/2); lacZ/alkaline phosphatase (Z/AP); MAPK kinase (MEK); MAPK kinase kinase (MEKK); mouse lung capillary endothelial cell (MLCEC); myocyte enhancer factor-2 (MEF2); polyinosinic-polycytidylic acid (pIpC).

The microRNA (miRNA) processing enzyme Dicer1 is required for zygotic and embryonic development, but the early embryonic lethality of Dicer1 null alleles in mice has limited our ability to address the role of Dicer1 in normal mouse growth and development. To address this question, we used a mouse mutant with a hypomorphic Dicer1 allele (Dicer d/d ) and found that Dicer1 deficiency resulted in female infertility. This defect in female Dicer d/d mice was caused by corpus luteum (CL) insufficiency and resulted, at least in part, from the impaired growth of new capillary vessels in the ovary. We found that the impaired CL angiogenesis in Dicer d/d mice was associated with a lack of miR17-5p and let7b, 2 miRNAs that participate in angiogenesis by regulating the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase 1. Furthermore, injection of miR17-5p and let7b into the ovaries of Dicer d/d mice partially normalized tissue inhibitor of metalloproteinase 1 expression and CL angiogenesis. Our data indicate that the development and function of the ovarian CL is a physiological process that appears to be regulated by miRNAs and requires Dicer1 function.

Mitogen-activated protein (MAP) kinase cascades play a central role in mediating extracellular stimuli-induced intracellular signaling during cell activation. The fourth and least studied mammalian MAP kinase pathway, big MAP kinase 1 (BMK1), also known as extracellular signal regulated kinase 5 (ERK5), is activated in response to growth factors and stress. Activation of this signaling pathway has been implicated not only in physiological functions such as cell survival, proliferation and differentiation but also in pathological processes such as carcinogenesis, cardiac hypertrophy and atherosclerosis. In recent years a series of gene-targeted mice lacking components within the BMK1 cascade have been generated, which have enabled us to investigate the role of the BMK1 pathway within different tissues. Analyses of these knockout mice have led to major discoveries in the role of BMK1 signaling in angiogenesis and in cardiac development. Moreover, studies using conditional BMK1 knockout mice, which circumvent the early embryonic lethality of BMK1 knockouts, have unveiled the importance of BMK1 in endothelial survival and maintenance of vascular integrity during adulthood. Here we summarize current understanding of the function of BMK1, as well as include new data generated from a series of tissue-specific BMK1 knockout mice in an attempt to dissect the role of the BMK1 pathway in various cell types in animals.

No abstract

Big mitogen-activated protein (MAP) kinase (BMK1), also known as ERK5, is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions. Previous studies have identified MEK5 as a cellular kinase directly regulating BMK1 activity; however, signaling molecules that directly regulate MEK5 activity have not yet been defined. Through utilization of a yeast two-hybrid screen, we have identified MEKK3 as a molecule that physically interacts with MEK5. This interaction appears to take place in mammalian cells as evidenced by the fact that cellular MEK5 and MEKK3 co-immunoprecipitate. In addition, we show that a dominant active form of MEKK3 stimulates BMK1 activity through MEK5. Moreover, we demonstrate that MEKK3 activity is required for growth factor mediated cellular activation of endogenous BMK1. Taken together, these results identify MEKK3 as a kinase that regulates the activity of MEK5 and BMK1 during growth factor-induced cellular stimulation.The mitogen-activated protein (MAP) 1 kinase cascades represent a primary mechanism by which cells transduce intracellular signals (1-3). These kinase cascades display a high degree of evolutionary conservation, as evidenced in a variety of eukaryotes ranging from yeast to mammals (2, 4). Three sequentially activated kinases make up the core of the MAP kinase module: a MAP kinase kinase kinase, or MEKK; a MAP kinase kinase, or MEK; and a MAP kinase (5, 6). In addition to delivering signals from extracellular stimuli to intended effectors, these kinase modules harmonize incoming signals from parallel signaling pathways and provide signal amplification as well as biological specificity. To date, four separate MAP kinases have been identified in mammalian cells and are known as ERK, JNK/SAPK, p38, and BMK1/ERK5 (7-13).BMK1/ERK5 represents the newest member of the mammalian MAP kinase family and was independently cloned by our laboratory and another group using different experimental approaches (7,11). We have previously demonstrated that BMK1 is activated by growth factors, oxidative stress, and hyperosmolar conditions (8,9,14). Upon activation, BMK1 stimulates the activity of myocyte enhancer factor 2C (MEF2C), a transcription factor that induces the expression of the proto-oncogene c-jun (9). Through the use of a dominant negative form of BMK1, we have demonstrated that BMK1 is required for growth factor-induced cell proliferation and cell cycle progression (8). Using the yeast two-hybrid system, MEK5 was identified by Zhou et al. (11) as the molecule responsible for regulating BMK1 activity. Subsequently, we have determined that MEK5 specifically activates BMK1 but not other mammalian MAP kinases in vivo (9). In addition, we have shown that MEK5 activity is required for the activation of BMK1 induced by extracellular stimuli (8, 9).The upstream kinase responsible for regulating MEK5 activity within the BMK1 signaling module has not yet been reported. In this regard, studies by English et a...

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.Genetic and biochemical studies have identified the mitogenactivated protein (MAP) 1 kinases as central intracellular molecules that deliver signals from activated cell surface receptors to downstream regulatory proteins. These MAP kinases have been conserved in all eukaryotes, ranging from yeast to mammals, and have a universal role in controlling cell growth through the regulation of cell cycle progression (1-6). The rate of cell cycle progression is tightly regulated by both growth factors and stress-related stimuli, and MAP kinases deliver and integrate both types of these extracellular signals to the cell cycle machinery by modulating the phosphorylation state of intracellular proteins. The MAP kinases ERK1/2, JNK1, and p38 control cell cycle progression by regulating either the expression or the activity of key molecules required for G 1 to S phase transition. We have previously demonstrated that BMK1/ERK5, the newest member of the MAP kinase family (7-11), is required for growth factor-induced cell proliferation and cell cycle progression (10). Although we have established that the activity of BMK1 is required for the growth factormediated entry of cells into the S phase of the cell cycle (10), the downstream effector(s) of this process have not yet been reported.To investigate the mechanism by which BMK1 mediates the entry of cells into S phase, this kinase was used as bait in a yeast two-hybrid screening of a cDNA library. Here we report the identification of serum-and glucocorticoid-inducible protein kinase (SGK) as a molecule that physically interacts with BMK1. SGK is a serine/threonine protein kinase with significant sequence homology throughout its catalytic domain with protein kinase B, ribosomal protein S6 kinase, cAMP-dependent protein kinase, and members of the protein kinase C family (12). A variety of stimuli, including glucocorticoids, hydrogen peroxide, hyperosmotic stress, serum, and insulin-like growth factor, have been shown to induce both the cellular expression and kinase activity of SGK (12-16). Similar to BMK1, the activity of SGK is closely linked to the G 1 /S transition of the cell cycle (17). Here, we show that BMK1 activates SGK as a result of growth factor-induced cellular activation through the phosphorylation of serine 78. Moreover, we demonstrate that the BMK1-mediated phosphorylation of SGK is critical fo...

Although big mitogen-activated protein kinase 1 (BMK1) has been shown to be critical for embryonic angiogenesis, the role of BMK1 in tumor-associated neovascularization is poorly understood. Exogenous tumors were established in BMK1 +/+ , BMK1 flox/+ , or BMK1 flox/flox mice carrying the Mx1-Cre transgene. Induced deletion of host BMK1 gene significantly reduced the volumes of B16F10 and LL/2 tumor xenografts in BMK1 flox/flox mice by 63% and 72%, respectively. Examining the tumors in these induced BMK1-knockout animals showed a significant decrease in vascular density. Localized reexpression of BMK1 in BMK1-knockout mice by administration of adenovirus encoding BMK1 restored tumor growth and angiogenesis to the levels observed in wild-type mice. These observations were further supported by in vivo Matrigel plug assays in which vascular endothelial growth factor-and basic fibroblast growth factor-induced neovacularization was impaired by removing BMK1. Through screening with the Pepchip microarray, we discovered that in BMK1-knockout endothelial cells, phosphorylation of ribosomal protein S6 (rpS6) at Ser235/ 236 was mostly abrogated, and this BMK1-dependent phosphorylation required the activity of p90 ribosomal S6 kinase (RSK). Immunofluorescent analysis of tumor vasculature from BMK1-knockout and control animals revealed a strong correlation between the presence of BMK1 and the phosphorylation of rpS6 in tumor-associated endothelial cells of blood vessels. As both RSK and rpS6 are known to be important for cell proliferation and survival, which are critical endothelial cell functions during neovascularization, these findings suggest that the BMK1 pathway is crucial for tumor-associated angiogenesis through its role in the regulation of the RSK-rpS6 signaling module. (Cancer Res 2005; 65(17): 7699-706)

No abstract