p53 independent,region‐specific epithelial apoptosis is induced in the rat epididymis by deprivation of luminal factors

Luminal testicular factors are known to be important for the regulation of the epididymal epithelium. The present study was undertaken to test the hypothesis that complete deprivation of luminal factors by efferent duct ligation (EDL) would induce apoptosis in the epididymal epithelium, as does removal of trophic factors from other cell types. Additionally, experiments were performed to determine whether the apoptosis detected was p53 dependent or independent. Apoptosis detection was by terminal deoxynucleotidyl‐mediated deoxyuridine triphosphate‐biotin nick‐end labeling and by DNA fragmentation studies. EDL caused loss of testicular luminal contribution in zone 1A of the rat epididymis (proximal initial segment) within 6 hr and induced epithelial apoptosis within 12 hr of the efferent duct obstruction. The wave of apoptosis in zone 1A was completed by three days after EDL and was followed by a much smaller wave in zone 1B which peaked three days after EDL. Significant apoptosis was not detected in any epididymal region distal to the initial segment for periods as long as 15 days after EDL. p53, a key apoptotic‐pathway molecule in many tissues and conditions was tested by immunohistochemical and Western blot techniques and was not upregulated in the initial segment epithelium within the time cells were undergoing apoptosis and well before the wave of apoptosis was complete. It was concluded that epithelial apoptosis in the initial segment of the rat epididymis is induced by deprivation of luminal testicular factors, is localized to the proximal and middle initial segment, and is p53 independent. Mol. Reprod. Dev. 53:188–197, 1999. © 1999 Wiley‐Liss, Inc.     

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis

The Ras/mitogen-activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual-specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal-regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium-induced colitis, as opposed to wild-type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc ^Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage-independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.     

In vitro treatment of human monocytes/macrophages with myristoylated recombinant Nef of human immunodeficiency virus type 1 leads to the activation of mitogen-activated protein kinases, IkappaB kinases, and interferon regulatory factor 3 and to the release of beta interferon

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-kappaB and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the alpha and beta subunits of the IkappaB kinase complex and of JNK, ERK1/2, and p38 mitogen-activated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.     

瘦素对卵巢癌细胞 SKOV3 增殖及凋亡的影响

目的:探讨瘦素对人卵巢癌SKOV3细胞增殖及凋亡的影响及其作用机制。方法:用不同浓度的瘦素(0、50、100、200 ng/m L)处理人卵巢癌SKOV3细胞48 h后,采用MTT法检细胞的生长;以血清饥饿诱导细胞凋亡,同时给予瘦素刺激,Annexin V/PI双染法检测细胞凋亡的变化;western blotting分析p21、cyclin D1、Bcl-2、Bax蛋白的表达水平和ERK1/2通路的活化情况。结果:瘦素以剂量依赖性的方式促进人卵巢癌SKOV3细胞的增殖,同时抑制血清饥饿诱导的细胞凋亡。瘦素处理可下调p21和上调cyclin D1的表达,抑制促凋亡分子Bax的表达和上调抗凋亡分子Bcl-2的表达。瘦素可诱导细胞中ERK1/2通路的活化,其抑制剂PD98059可明显抑制瘦素诱导的促细胞增殖和抗凋亡作用,同时伴随有cyclin D1、Bcl-2蛋白表达的下调和Bax的上调。结论:瘦素可能通过活化ERK1/2通路调节细胞有丝分裂进程,进而促进卵巢癌细胞的增殖;同时通过调节凋亡相关蛋白Bcl-2和Bax的表达抑制卵巢癌细胞的凋亡。     

Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only protein,Bim

Both the ERK and phosphatidylinositol 3'-kinase (PI3K) signaling pathways can protect cells from apoptosis following withdrawal of survival factors. We have previously shown that the ERK1/2 pathway acts independently of PI3K to block expression of the BH3-only protein, BimEL, and prevent serum withdrawal-induced cell death, although the precise mechanism by which ERK reduced BimEL levels was unclear. By comparing Bim mRNA and Bim protein, expression we now show that the rapid expression of BimEL following serum withdrawal cannot be accounted for simply by increases in mRNA following inhibition of PI3K. In cells maintained in serum BimEL is a phosphoprotein. We show that activation of the ERK1/2 pathway is both necessary and sufficient to promote BimEL phosphorylation and that this leads to a substantial increase in turnover of the BimEL protein. ERK1/2-dependent degradation of BimEL proceeds via the proteasome pathway because it is blocked by proteasome inhibitors and is defective at the restrictive temperature in cells with a temperature-sensitive mutation in the E1 component of the ubiquitin-conjugating system. Finally, co-transfection of BimEL and FLAG-ubiquitin causes the accumulation of polyubiquitinated forms of Bim, and this requires the ERK1/2 pathway. Our findings provide new insights into the regulation of Bim and the role of the ERK pathway in cell survival.     

Expression of CNTF/LIF-receptor components and activation of STAT3 signaling in axotomized facial motoneurons: evidence for a sequential postlesional function of the cytokines

Several lines of evidence suggest that ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are important for the survival and regeneration of axotomized motoneurons. To investigate the role of CNTF/LIF signaling in regenerative responses of motoneurons, we studied the expression of the three receptor components, CNTF receptor alpha (CNTFRalpha), LIF receptor beta (LIFRbeta), and gp130, and the activation of the STAT3 signal transduction pathway in the rat facial nucleus following peripheral nerve transection. As shown by in situ hybridization and immunoblotting, axotomy resulted in a rapid down-regulation of CNTFRalpha mRNA expression within 24 h and a concomitant massive up-regulation of LIFRbeta mRNA and protein in the lesioned motoneurons. The altered mRNA levels were maintained for 3 weeks but had returned back to control levels by 6 weeks postlesion after successful regeneration. In contrast, mRNA levels remained in the lesioned state during the 6-week period studied, when regeneration was prevented by nerve resection. Significant lesion-induced changes in gp130 mRNA levels were not detectable. Rapid (within 24 h) and sustained (for at least 5 days) activation of STAT3 in axotomized facial motoneurons was revealed by demonstrating the phosphorylation and nuclear translocation of the protein using immunocytochemistry and immunoblotting. In agreement with previous studies showing a complementary regulation of CNTF and LIF in the lesioned facial nerve, our observations on the postlesional regulation of CNTF/LIF receptor components in the facial nucleus indicate a direct and sequential action of the two neurotrophic proteins on axotomized facial motoneurons.     

Growth hormone-induced differential desensitization of STAT5, ERK,and Akt phosphorylation

Secretion of growth hormone (GH) in adult male rats is characterized by high peak and undetectable trough levels, both of which are required for male-specific pattern of liver gene expression and GH-induced phosphorylation of STAT5. The present study suggests that regulation of GH receptor (GHR) levels in rat hepatoma cells by repeated GH stimulation determines GH responsiveness via the JAK2/STAT5 pathway. A short exposure to GH rapidly reduced GHR levels which resulted in an equal desensitization of the JAK2/STAT5 pathway. Recovery of GH-induced STAT5 phosphorylation correlated with the time-dependent recovery of GHR levels during incubation in the absence of GH. Acute GH also induced phosphorylation of ERK1/2 and Akt, and this induction was also inhibited by prior exposure to GH. However, unlike the JAK2/STAT5 pathway, the effect of GH to activate the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways did not recover following prolonged incubation in the absence of GH. Thus, GH administration desensitizes the JAK2/STAT5 pathway, possibly because of down-regulation of GHR, whereas an additional post-receptor mechanism is required for the prolonged refractoriness of the MEK/ERK and phosphatidylinositol 3-kinase/Akt pathways toward a second GH stimulation. Our study suggests that both receptor and post-receptor mechanisms are important in GH-induced homologous desensitization.     

Follicle-stimulating hormone-induced aromatase in immature rat Sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway

Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.     

Leptin stimulates tissue inhibitor of metalloproteinase-1 in human hepatic stellate cells: respective roles of the JAK/STAT and JAK-mediated H2O2-dependant MAPK pathways

Leptin is recognized as a profibrogenic hormone in the liver, but the mechanisms involved have not been clarified. The tissue inhibitor of metalloproteinase (TIMP)-1, which acts through inhibition of collagen degradation, is synthesized by activated hepatic stellate cells (HSC) in response to fibrogenic substances. The capacity of leptin to induce TIMP-1 and its signaling molecules were investigated in a human HSC cell line, LX-2. Leptin stimulated TIMP-1 protein, mRNA, and promoter activity. JAK1 and -2, as well as STAT3 and -5, were activated. After leptin, there was increased expression of tyrosine 1141-phosphorylated leptin receptor, which may contribute to STAT3 activation. AG 490, a JAK inhibitor, blocked JAK phosphorylation with concomitant inhibition of STAT activation, TIMP-1 mRNA expression, and promoter activity. Leptin also induced an oxidative stress, which was inhibited by AG 490, indicating a JAK mediation process. ERK1/2 MAPK and p38 were activated, which was prevented by catalase, indicating an H2O2-dependent mechanism. Catalase treatment resulted in total suppression of TIMP-1 mRNA expression and promoter activity. SB203580, a p38 inhibitor, prevented p38 activation and reduced TIMP-1 message half-life with down-regulation of TIMP-1 mRNA. These changes were reproduced by overexpression of the dominant negative p38alpha and p38beta mutants. PD098059, an ERK1/2 inhibitor, opposed ERK1/2 activation and TIMP-1 promoter activity, leading to TIMP-1 mRNA down-regulation. Thus, leptin has a direct action on liver fibrogenesis by stimulating TIMP-1 production in activated HSC. This process appears to be mediated by the JAK/STAT pathway via the leptin receptor long form and the H2O2-dependent p38 and ERK1/2 pathways via activated JAK.     

Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1BN Rats

We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1^BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1^BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1^BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1^BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1^BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.     

Dual-specificity phosphatases regulate mitogen-activated protein kinase signaling in adipocytes in response to inflammatory stress

Obesity is a strong predictor of heart disease, insulin resistance, and type II diabetes. Chronic, low-grade inflammation links obesity and insulin resistance through mitogen-activated protein kinase (MAPK) signaling pathways. Upstream kinases activate MAPK signaling, while MAPK-specific dual-specificity phosphatases (DUSPs) act as key modulators and controllers of MAPK deactivation (i.e. dephosphorylation). Using tumor necrosis factor α (TNFα) in 3 T3-L1 adipocytes as a model of inflammation, we report that TNFα-mediated induction of Dusp1, Dusp8 and Dusp16 modulated the transient regulation of MAPK (i.e., ERK, JNK, and p38) phosphorylation and subsequent inflammatory gene expression. All three MAPKs examined were phosphorylated in preadipocytes and adipocytes in response to TNFα, where signaling magnitude and duration were phenotype-specific. Moreover, TNFα increased mRNA abundance of DUSPs in preadipocytes and adipocytes in a phenotype-specific manner, concomitant with dephosphorylation of MAPKs. RNA interference (RNAi)-mediated knockdown of Dusp1, Dusp8 and Dusp16 increased signaling magnitude and duration of ERK, JNK, and p38 that subsequently resulted in significant increases in MAPK-dependent inflammatory gene expression of MCP-1, IL-6, and Cox-2 in response to TNFα. This study highlights important roles for DUSPs as integral components of MAPK signaling and adipocyte inflammatory gene expression.     

RanBPM Is an Inhibitor of ERK Signaling

Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein of yet unknown function. We have previously shown that RanBPM inhibits expression of the anti-apoptotic factor Bcl-2 and promotes apoptosis induced by DNA damage. Here we show that the effects of RanBPM on Bcl-2 expression occur through a regulation of the ERK signaling pathway. Transient and stable down-regulation of RanBPM stimulated ERK phosphorylation, leading to Bcl-2 up-regulation, while re-expression of RanBPM reversed these effects. RanBPM was found to inhibit MEK and ERK activation induced by ectopic expression of active RasV12. Activation of ERK by active c-Raf was also prevented by RanBPM. Expression of RanBPM correlated with a marked decrease in the protein levels of ectopically expressed active c-Raf and also affected the expression of endogenous c-Raf. RanBPM formed a complex with both active c-Raf, consisting of the C-terminal kinase domain, and endogenous c-Raf in mammalian cells. In addition, RanBPM was found to decrease the binding of Hsp90 to c-Raf. Finally, we show that loss of RanBPM expression confers increased cell proliferation and cell migration properties to HEK293 cells. Altogether, these findings establish RanBPM as a novel inhibitor of the ERK pathway through an interaction with the c-Raf complex and a regulation of c-Raf stability, and provide evidence that RanBPM loss of expression results in constitutive activation of the ERK pathway and promotes cellular events leading to cellular transformation and tumorigenesis.