The 41-kDa and 43-kDa mitogen-activated protein (MAP) kinases play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAP kinase cascade, which includes MAP kinase kinase (MEK) and Raf-1. As aberrant activation of signal transducing molecules such as Ras and Raf-1 has been linked with cancer, we examined whether constitutive activation of the 41-/43-kDa MAP kinases is associated with the neoplastic phenotype of 138 tumor cell lines and 102 primary tumors derived from various human organs. Constitutive activation of the MAP kinases was observed in 50 tumor cell lines (36.2%) in a rather tissue-speci®c manner: cell lines derived from pancreas, colon, lung, ovary and kidney showed especially high frequencies with a high degree of MAP kinase activation, while those derived from brain, esophagus, stomach, liver and of hematopoietic origin showed low frequencies with a limited degree of MAP kinase activation. We also detected constitutive activation of the 41-/43-kDa MAP kinases in a relatively large number of primary human tumors derived from kidney, colon and lung tissues but not from liver tissue. Many tumor cells, in which point mutations of ras genes were detected, showed constitutive activation of MAP kinases, however, there were also many exceptions to this observation. In contrast, the activation of the 41-/43-kDa MAP kinases was accompanied by the activation of Raf-1 in the majority of tumor cells and was completely associated with the activation of MEK and p90 rsk in all the tumor cells examined. These results suggest that the constitutive activation of 41-/43-kDa MAP kinases in tumor cells is not due to the disorder of MAP kinases themselves, but is due to the disorder of Raf-1, Ras, or some other signaling molecules upstream of Ras.

The extracellular signal-regulated kinase (ERK) signaling pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, differentiation and motility. This pathway is often up-regulated in human tumors and as such represents an attractive target for the development of anticancer drugs. Because of its multiple roles in the acquisition of a complex malignant phenotype, specific blockade of the ERK pathway is expected to result in not only an anti-proliferative effect but also in anti-metastatic and anti-angiogenic effects in tumor cells. Recently potent small-molecule inhibitors targeting the components of the ERK pathway have been developed. Among them, BAY 43-9006 (Raf inhibitor), and PD184352, PD0325901 and ARRY-142886 (MEK1/2 inhibitors) have reached the clinical trial stage. We briefly discuss the possibility that combination of ERK pathway inhibitors (cytostatic agents) and conventional anticancer drugs (cytotoxic agents) provides an excellent basis for the development of new chemotherapeutic strategies against cancer.

Bone morphogenetic proteins (BMPs), 1 members of the transforming growth factor-␤ superfamily, were originally identified by their novel activity to induce cartilage and bone formation in ectopic extraskeletal sites in vivo (1, 2). A number of studies have demonstrated that BMPs play essential roles in bone formation and bone cell differentiation; e.g. BMPs stimulate proteoglycan synthesis, alkaline phosphatase activity, collagen synthesis, and osteocalcin expression in chondroblasts/ osteoblasts (3-5). However, expression of BMPs (6 -8) and their receptors (9) in many tissues other than bone suggests that they are also involved in the regulation of many biological processes unrelated to bone formation. In accordance with this possibility, increasing evidence suggests a regulatory role for BMPs in early vertebrate embryogenesis such as in mesoderm induction, limb development, and hematopoietic formation (10 -14). Moreover, BMPs have drawn attention as possible regulators of central nervous system development; BMPs are expressed in multiple central nervous system regions throughout development (15)(16)(17). In this respect, we have recently demonstrated that BMP-2 acts as a neurotrophic factor; BMP-2 induces the neuronal differentiation of rat pheochromocytoma PC12 cells (18) and also promotes the survival and differentiation of striatal GABAergic neurons in culture (19).BMPs transduce their signals by binding to two different types of serine/threonine kinase receptor, type I and type II (20 -22). Upon ligand binding, type I and type II receptors form heteromeric receptor complexes. Then type I receptors are phosphorylated by type II receptors, and subsequent activation of the catalytic activity of type I receptor kinase is essential for BMP signaling. Although phosphorylation of Smad proteins by the activated type I receptor kinase was suggested to play an important role in the mediation of BMP signaling (20 -22), the precise cytoplasmic signaling pathway for BMPs through which it elicits its effects on the regulation of wide variety of biological processes remains largely unknown.We have recently shown that BMP-2 has the capacity to induce the neuronal differentiation of PC12 cells. Unlike NGF, however, BMP-2 did not induce the activation of MEK or 41-/ 43-kDa MAP kinases (ERK2 and ERK1, respectively) in these cells (18). MEK and ERK1/2 constitute a protein kinase cascade (the ERK cascade), which is one of the major cytoplasmic signaling pathways commonly activated in a wide variety of cells by diverse extracellular stimuli (23)(24)(25)(26). Activation of the cascade elicits a wide array of physiological responses such as cell division, differentiation, and secretion (23-26), and it has been reported that the activation of the cascade is necessary and sufficient for PC12 cell neuronal differentiation (27,28). However, our previous findings, which showed a clear distinction between the requirement for the activation of MEK and ERK1/2 and the ability of BMP-2 to induce PC12 cell neuronal differentiation (18), hav...

Tumor necrosis factor-␣ (TNF-␣), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-␣, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-␣ on the Rho pathway in tubular cells (LLC-PK 1 and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-␣-induced alterations of paracellular permeability. We show that TNF-␣ induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-␣-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-␣-induced activation of these proteins. Importantly TNF-␣ enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-␣-induced paracellular permeability increase was absent in LLC-PK 1 cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-␣-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.

The expression of mitogen-activated protein kinases (MAPK) in DBA/2-pcy/pcy (pcy) mice, a murine model of polycystic kidney disease was investigated. Proliferating cell nuclear antigen-positive cells were recognized in cyst epithelium from embryonic day 14.5 to 25 wk of age. Extracellular signal-regulated kinase (ERK) was expressed in the renal tubules of control and pcy mice, but stronger immunostaining was observed in cyst epithelium. Phosphorylated ERK was detected only in pcy mice and was localized predominantly in the cysts. p38 MAPK (p38) was no longer expressed after birth in controls but was detected in the cyst epithelium and in occasional tubular cells of pcy mice at all stages examined. c-Jun N-terminal kinase (JNK) was expressed in all tubular segments of controls after neonatal day 7, whereas in pcy kidneys, tubules became positive for JNK after 8 wk, and the cysts expressed little JNK. Administration of an oral MAP/ERK kinase inhibitor, PD184352, 400 mg/kg per d, to 10-wk-old pcy mice daily for the first week and then every third day for 6 additional weeks significantly decreased BP, kidney weight, serum creatinine level, and water intake and significantly increased urine osmolality. The cystic index and expression of phosphorylated ERK and ERK were significantly lower in PD184352-treated pcy mice. These results demonstrate that the expression of MAPK is dysregulated in cyst epithelium and that inhibition of ERK slowed the progression of renal disease in pcy mice.