phospho-Jun N-terminal Kinase Research Articles

SZ168 treats LPS-induced acute lung injury by inhibiting the activation of NF-κB and MAPKs pathways

BackgroundThis study aimed to elucidate the effect and underlying molecular mechanisms of SZ168 (Podoplanin (PDPN) monoclonal antibody) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice and LPS-induced MH-S cells. MethodsThe survival rate was calculated by recording the death of mice in each group. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of interleukin (IL)− 6 and tumor necrosis factor-alpha (TNF-α) in blood and bronchoalveolar lavage fluid (BALF) of mice. Hematoxylin-eosin (H&E) staining were performed to evaluate the pathological changes in pulmonary tissues. Additionally, the phagocytosis of cells was tested by flow cytometry, and the expression levels of caveolin-1 (CAV-1) and Occludin proteins in lung tissue and the expression of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway-related proteins in MH-S cells were determined by western blot. ResultsSZ168 significantly improved the survival rate of ALI mice. Briefly speaking, SZ168 protected pulmonary vascular permeability, reduced the level of pro-inflammatory cytokines, improved the pathological changes of lung tissue, reduced the infiltration of inflammatory cells, increased CAV-1 and Occludin protein expression, and then effectively relieved lung injury. In addition, SZ168 could significantly reduce the phagocytic ability of LPS-induced MH-S cells and inhibit the expression of hospho-extracellular regulated protein kinases (p-ERK), Phospho-Jun N-terminal kinase (p-JNK), Phospho-NF-κB p65 (p-p65) and Phospho-IkappaB-alpha (p-IκBα). ConclusionSZ168 can treat ALI by inhibiting the activation of NF-κB and MAPK signaling pathways and restoring tight junction protein expression.

Nicotinamide adenine dinucleotide attenuates acetaminophen-induced acute liver injury via activation of PARP1, Sirt1, and Nrf2 in mice.

The aim of this study was to investigate the protective effect of nicotinamide adenine dinucleotide (NAD+) against acute liver injury (ALI) induced by acetaminophen (APAP) overdose in mice. First, serum transaminases were used to assess the protective effect of NAD+, and the data revealed that NAD+ mitigated the APAP-induced ALI in a dose-dependent manner. Then, we performed hematoxylin-eosin staining of liver tissues and found that NAD+ alleviated the abnormalities of histopathology. Meanwhile, increase in the malondialdehyde content and decrease in glutathione, superoxide dismutase (SOD), and glutathione peroxidase were identified in the APAP group, which were partially prevented by the NAD+ pretreatment. Moreover, compared with the mice treated with APAP only, the expression of poly ADP-ribose polymerase 1 (PARP1), Sirtuin1 (Sirt1), SOD2, nuclear factor erythroid 2-related factor 2 (Nrf2), and hemoxygenase-1 was upregulated, while Kelch-like ECH-associated protein 1 and histone H2AX phosphorylated on Ser-139 were downregulated by NAD+ in NAD+ + APAP group. Conversely, NAD+ could not correct the elevated expression of phospho-Jun N-terminal kinase and phospho-extracellular signal-regulated kinase induced by APAP. Taken together, these findings suggest that NAD+ confers an anti-ALI effect to enhance the expression of PARP1 and Sirt1, and to simultaneously stimulate the Nrf2 anti-oxidant signaling pathway.

Corydalis decumbens Can Exert Analgesic Effects in a Mouse Neuropathic Pain Model by Modulating MAPK Signaling.

Objectives This study is aimed at investigating the analgesic effect of the administration of Corydalis decumbens (CD) in a mouse model of postherpetic neuralgia (PHN) and at elucidating its mechanism of analgesic action. Methods Adult Kunming (KM) mice were randomly divided into control, CD, and vehicle-treated groups. Neuropathic pain was induced with a single intraperitoneal injection of resiniferatoxin (RTX). Thermal hyperalgesia was assessed with a hot/cold plate test, and mechanical allodynia was evaluated using von Frey filaments. The activation states of astrocytes, microglia, and the mitogen-activated protein kinase (MAPK) pathway in the spinal cord were determined by immunofluorescence staining and Western blot analysis of Iba-1, GFAP, phospho-p38, and phospho-Jun N-terminal kinase (JNK). Results RTX diminished thermal sensitivity and gradually increased sensitivity to tactile stimulation. The expression of Iba-1, GFAP, phospho-p38 MAPK, and phospho-JNK was upregulated in the RTX-induced postherpetic neuralgia mouse model. Systemic treatment with CD significantly ameliorated thermal sensitivity and mechanical hyperalgesia and was accompanied by a reduction in the expression of Iba-1 and GFAP and reduced phosphorylation of p38 and JNK. Conclusions This study suggests that CD is effective at ameliorating mechanical hyperalgesia in PHN mice and that its mechanism of action may involve modulation of MAPK phosphorylation and glial cell activation. Thus, CD may be a promising alternative therapy for PHN.

Role of genistein on the yield, structure and immunomodulatory activity of Monascus exopolysaccharides.

Manipulating the structures, physicochemical properties, and monosaccharide compositions of exopolysaccharides (EPS) isolated from microorganisms has been reported to enhance their biological activities. Hence, the aim of this work was to examine the effects of genistein addition during fermentation on the amount, physicochemical properties, and immunomodulatory activity of EPS secreted by M. purpureus. Results showed that genistein addition significantly increased M. purpureus biomass and EPS yield to 2.42 g L-1 and 6.08 g L-1, respectively, and affected the physicochemical properties and structures of EPS. Furthermore, EPS produced by genistein-treated M. purpureus (G-EMP) improved the immunomodulatory activity of RAW264.7 macrophages by increasing the secretion of nitric oxide and cytokines. Moreover, phospho-Jun N-terminal kinase (p-JNK), phospho-extracellular regulated protein kinase (p-ERK), phospho-p38 (p-p38) mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) phospho-p65 (p65) proteins were remarkably upregulated by G-EMP stimulation, blocking Toll-like receptor 4 (TLR4) that dramatically reduced the pinocytic and phagocytic capacities. Overall, these findings provide potential rationales for the application of genistein in improving the EPS yield of M. purpureus.

Beta-Amyrin Modulates P38 MAPK and Jnk Pathway to Inhibit Cell Proliferation and Induce ROS-mediated Apoptosis in HeLa Cells

It is aimed to investigate the effect of ?-amyrin on p38 mitogen-activated protein kinase and Jun N-terminal kinase pathways and apoptosis in HeLa cells. HeLa treated cells were divided into 6 groups, group IHeLa untreated cells as control, group II- dimethyl sulfoxide serve as vehicle control, group III- cisplatin as standard drug, group IV- ?-amyrin-treated HeLa cells, group V- cells were pretreated with 100 ?m N-acetyl-L-cystein for 1 hour and then treated with cisplatin and group VI- cells were pretreated with 100 ?m N-acetyl-L-cystein for 1 hour and then treated with ?-amyrin. The antiproliferative effect was measured using the MTT assay. Genotoxic effects were studied using micronucleus assay. Total reactive oxygen species, nitric oxide and caspase 3 level were determined on a spectrofluorimeter and colorimeter. Protein expression was analyzed by immunoblotting. ?-Amyrin (10-200 ?m) and cisplatin (0.01-100 ?m) had an inhibitory effect on the proliferation of cancer cells in a dose-dependent manner, with the IC50 values at 100 ?m and 10 ?m for ?-amyrin and cisplatin, respectively. Western blot analysis revealed expressions of apoptotic pathway related proteins, Bcl-2, caspase-3, caspase-9, phospho-p38 mitogen-activated protein kinase and phospho-Jun N-terminal kinase, growth arrest and deoxyribonucleic acid-damage-inducible, beta in all groups. Genotoxic effects were observed after treatment with ?-amyrin as well as with cisplatin. It was observed that HeLa cells showed significant elevation of total reactive oxygen species after ?-amyrin treatment. Protein expression analysis showed that the ?-amyrin upregulated phospho-p38 mitogenactivated protein kinase, phospho-Jun N-terminal kinase and growth arrest and deoxyribonucleic aciddamage- inducible, beta on HeLa cells. Increased phospho-Jun N-terminal kinase directly activated caspases and decreased Bcl-2 in HeLa cells. These results indicated that ?-amyrin induced the apoptosis through reactive oxygen species-mediated mechanism by activating p38 mitogen-activated protein kinase and Jun N-terminal kinase through transcriptional factor, GADD45?. In turn, activated Jun N-terminal kinase directly activated caspase-9 and caspase-3 and destined the HeLa cells to apoptosis.

Chlorogenic acid protects against aluminum toxicity via MAPK/Akt signaling pathway in murine RAW264.7 macrophages

Aluminum (Al), which may bring about damage to the macrophages, has been implicated in the development of immunological diseases. It has been reported that chlorogenic acid (CGA, 5‑caffeoylquinic acid, chemical formula: C16H18O9) is a natural antioxidant and chelating agent with the capacity against Al (III)-induced biotoxicity. The present study was carried out to investigate whether CGA could reduce AlCl3-induced cellular damage in RAW264.7 cells. After treatment with AlCl3, the inhibition rate of viability and phagocytic activity of RAW264.7 cells was 54.5% and 27.6%, respectively. Administration of CGA significantly improved the integrity and phagocytic activity, and attenuated the accumulation of intracellular Al(III) level and oxidative stress in Al(III)-treated cells. Furthermore, CGA significantly inhibited Al(III)-induced increase of phospho-Jun N-terminal kinase (p-JNK), a pro-apoptotic Bcl-2 family protein (Bad), cytochrome c and decrease of extracellular regulated protein kinases (ERK1/2), protein kinase B (Akt) protein expressions. These results showed that CGA has a protective effect against Al(III)-induced cytotoxicity through mitogen-activated protein kinase (MAPK)/Akt-mediated caspase pathways in RAW264.7 cells.

The Hajdu Cheney Mutation Is a Determinant of B-Cell Allocation of the Splenic Marginal Zone

The neurogenic locus notch homolog protein (Notch)-2 receptor is a determinant of B-cell allocation, and gain-of-NOTCH2-function mutations are associated with Hajdu-Cheney syndrome (HCS), a disease presenting with osteoporosis and acro-osteolysis. We generated a mouse model reproducing the HCS mutation (Notch2HCS), and heterozygous global mutant mice displayed gain-of-Notch2 function. In the mutant spleen, the characteristic perifollicular rim marking the marginal zone (MZ), which is the interface between the nonlymphoid red pulp and the lymphoid white pulp, merged with components of the white pulp. As a consequence, the MZ of Notch2HCS mice occupied most of the splenic structure. To explore the mechanisms involved, lymphocyte populations from the bone marrow and spleen were harvested from heterozygous Notch2HCS mice and sex-matched control littermates and analyzed by flow cytometry. Notch2HCS mice had an increase in CD21/35highCD23- splenic MZ B cells of approximately fivefold and a proportional decrease in splenic follicular B cells (CD21/35intCD23+) at 1, 2, and 12 months of age. Western blot analysis revealed that Notch2HCS mutant splenocytes had increased phospho-Akt and phospho-Jun N-terminal kinase, and gene expression analysis of splenic CD19+ B cells demonstrated induction of Hes1 and Hes5 in Notch2HCS mutants. Anti-Notch2 antibodies decreased MZ B cells in control and Notch2HCS mice. In conclusion, Notch2HCS mutant mice have increased mature B cells in the MZ of the spleen.

Assessment of Protective Role of Multifunctional Dopamine Agonist D-512 Against Oxidative Stress Produced by Depletion of Glutathione in PC12 Cells: Implication in Neuroprotective Therapy for Parkinson's Disease.

Oxidative stress has been strongly implicated in the progression of Parkinson's disease (PD). Depletion of cytoplasmic glutathione levels is one of the indications of oxidative stress, which occur in the substantia nigra of PD patients at an early stage of the disease process. It has been shown that glutathione depletion causes the inhibition of mitochondrial complex I, thus affecting mitochondrial function leading to oxidative stress via production of reactive oxygen species. Studies were carried out to investigate the role of D-512, a potent multifunctional neuroprotective D2/D3 receptor agonist, in protecting dopaminergic PC12 cells treated with buthionine sulfoximine (BSO), an inhibitor of key enzyme in glutathione synthesis and 6-hydroxydopamine (6-OHDA), a widely used neurotoxin. D-512 was able to restore level of glutathione against BSO/6-OHDA-mediated glutathione depletion. D-512 also showed significant neuroprotection in PC12 cells against toxicity induced by combined treatment of BSO and 6-OHDA. Furthermore, D-512 was able to restore both phospho-extracellular signal-regulated kinase and phospho-Jun N-terminal kinase levels upon treatment with 6-OHDA providing an evidence on the possible mechanism of action for neuroprotection by modulating mitogen-activated protein kinases. We have further demonstrated the neuroprotective effects of D-512 against oxidative insult produced by BSO and 6-OHDA in PC12 cells.

Bortezomib inhibits gastric carcinoma HGC-27 cells through the phospho-Jun N-terminal kinase (p-JNK) pathway in vitro

The study is designed to explore the anticancer mechanism of Bortezomib. The effects of Bortezomib on the proliferation of human gastric cancer cells HGC-27 and expression levels of the phospho-Jun N-terminal kinase (p-JNK) pathway-related proteins in vitro were investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the Bortezomib significantly decreased the viability of HGC-27 cells and induced apoptosis. Western blot showed that the Bortezomib strongly increased the levels of p-JNK, caspase-3, PARP, and bax proteins while it increased the level of bcl-2. However, SP600125 can significantly decrease antitumour effects of Bortezomib in HGC-27 cells. It can be concluded that Bortezomib has significant inhibitory effects on the growth of HGC-27 cells. The effect may be achieved partly via upregulating JNK pathway and its down-stream apoptosis-related proteins. Therefore, Bortezomib may be beneficial in gastric carcinoma treatment.

Aqueous extract of Taxus Chinensis (Pilger) Rehd inhibits lung carcinoma A549 cells through the epidermal growth factor receptor/mitogen-activated protein kinase pathway in vitro and in vivo

ObjectiveTo explore the anticancer mechanism of aqueous extract of Taxus Chinensis (Pilger) Rehd (AETC). MethodsThe serum pharmacological method was used to avoid interference from administration of the crude medicinal herbs. Eight purebred New Zealand rabbits were used for preparation of serum containing various concentrations of AETC. Forty-eight Balb/c-nu mice were used for in vivo experiments. The effects of serum containing AETC on the proliferation of A549 cells and expression levels of the epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway-related proteins in vitro were investigated. Additionally, the effects on the growth of A549 xenografts in nude mice, and expression levels of the EGFR/MAPK pathway-related proteins in the xenografts, were investigated. Results3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the serum containing AETC significantly decreased the viability of A549 cells in a dose-dependent manner. Western blot showed that the serum containing various concentrations of AETC strongly reduced the levels of phospho-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinasel/2 (ERK1/2) while it increased the level of p-p38. However, no significant effects on the expression levels of JNK, ERK1/2, and p38 MAPK were found. In addition, an anticancer effect from AETC was observed in vivo in the Balb/c-nu mice bearing A549 xenografts. ConclusionAETC has significant effects on the growth of A549 xenografts and on the activity of the EGFR/MAPK pathway. Therefore, AETC may be beneficial in lung carcinoma treatment.

Schistosoma japonicum egg antigen up-regulates fibrogenesis and inhibits proliferation in primary hepatic stellate cells in a concentration-dependent manner

To investigate the effects of different concentrations of Schistosoma japonicum (S. japonicum) egg antigen on fibrogenesis and apoptosis in primary hepatic stellate cells (HSCs). A mouse model of schistosomiasis-associated liver fibrosis (SSLF) was established by infecting mice with schistosomal cercaria via the abdomen. HSCs were isolated from SSLF mice by discontinuous density gradient centrifugation, and their identity was confirmed by immunofluorescence double staining of α-smooth muscle actin (α-SMA) and desmin. The growth inhibitory effect and 50% inhibitory concentration (IC50) of S. japonicum egg antigen for primary HSCs (24 h) were determined using a cell counting kit-8 (CCK-8) assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMOL/LP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in HSCs in response to different concentrations of S. japonicum egg antigen were detected by Western blotting and real-time reverse transcription-polymerase chain reaction. The levels of phospho-P38 (P-P38), phospho-Jun N-terminal kinase (P-JNK) and phospho-Akt (P-AKT) in HSCs were detected by Western blotting. An SSLF mouse model was established, and primary HSCs were successfully isolated and cultured. S. japonicum egg antigen inhibited HSC proliferation in a concentration-dependent manner. The IC50 of the S. japonicum egg antigen was 244.53 ± 35.26 μg/mL. S. japonicum egg antigen enhanced α-SMA expression at both the mRNA and protein levels and enhanced TIMP-1 expression at the mRNA level in HSCs (P < 0.05), whereas the expression of MMOL/LP-9 was attenuated at both the mRNA and protein levels in a concentration-dependent manner (P < 0.05). A high concentration of S. japonicum egg antigen enhanced P-P38, P-JNK and P-AKT activation (P < 0.05). The changes in α-SMA and MMOL/LP-9 expression induced by S. japonicum egg antigen were closely correlated with P-P38 and P-JNK activation (P < 0.05). The attenuation of MMOL/LP-9 was also correlated with P-AKT activation (P < 0.05), but the increase in α-SMA expression was not. TIMP-1 expression was not correlated with P-P38, P-JNK or P-AKT activation. S. japonicum egg antigen promotes fibrogenesis, activates the P38/JNK mitogen-activated protein kinase and AKT/PI3K signaling pathways and inhibits proliferation in primary HSCs isolated from SSLF mice in a concentration-dependent manner.

Escharectomy and Allografting During Shock Stage Reduces Insulin Resistance Induced by Major Burn

Hyperglycemia and insulin resistance have long been recognized in severe burn patients. Early excision and grafting reduces cytokines and insulin resistance in burned rats. The authors hypothesized that early wound excision and grafting in patients would also reduce insulin resistance induced by major burn. Thirty-five adult surviving major burn patients (>40%TBSA burn) were recruited. The removal of dead devitalized tissue and allografting in escharectomy group was performed within 72 hours and in control group about 7 days after burn injury. The concentrations of plasma insulin, glucose, and cytokines were measured at 2 and 5 days postburn. Euglycemic-hyperinsulinemic glucose clamps were performed at 5 days after burn. The levels of phosphotyrosine, phosphoserine³¹² of insulin receptor substrate (IRS)-1, and phospho-jun N-terminal kinase (JNK) in muscle were analyzed with immunoprecipitation and Western blotting at 5 days postburn. Escharectomy and allografting during shock stage significantly reduced the levels of interleukin-6 and tumor necrosis factor-α, decreased the levels of phosphoserine³¹² and phospho-JNK, increased the level of phosphotyrosine of IRS-1, and further reduced insulin resistance at 5 days after thermal injury compared with delayed excision group. Escharectomy and allografting during shock stage seemed to have an immunomodulatory effect on the inflammatory mediators and further to reduce insulin resistance induced by major burns in patients by decreasing the phosphorylation of IRS-1 serine³¹² and JNK1/2.

Effects of Pituitary Adenylate Cyclase Activating Polypeptide on the Survival and Signal Transduction Pathways in Human Choriocarcinoma Cells

The pituitary adenylate cyclase activating polypeptide (PACAP) has several effects in endocrine and reproductive organs, including the placenta. PACAP is generally known as a survival-promoting peptide acting on divergent signal transduction pathways. However, its effects on the survival and signaling mechanisms of trophoblast cells are not known. In the present study we found that 1-h pretreatment with PACAP38 did not significantly influence the survival of JAR cytotrophoblast cells. However, the survival rate of cells exposed to oxidative stress or CoCl(2)-induced in vitro hypoxia showed a significant further decrease in PACAP-treated cells, implying that PACAP sensitizes the cells to these stressors. This was not observed in the case of lipopolysaccharide or ethanol treatment. Western blot data revealed that, in cells exposed to oxidative stress, PACAP treatment decreased phosphorylation of all extracellular signal-regulated kinase (ERK), phospho-jun N-terminal kinase (JNK), protein kinase B, p38 mitogen-activated protein kinase (MAPK), and phospho-glycogen synthase kinase (GSK) and the expression of bax. The overall effect seems to be a sensitizing effect in almost all examined pathways when oxidative stress was applied, which may explain the enhancing effect of PACAP on cell death in contrast to most other cell types examined so far. Our data show that the signaling mechanism of PACAP may be different in trophoblast cells to that observed in other cell lines.

Activation of c-Jun N-terminal kinase in A549 lung carcinoma cells by sodium dichromate: role of dissociation of apoptosis signal regulating kinase-1 from its physiological inhibitor thioredoxin.

Changes in the components of the Jun N-terminal kinase (JNK) signalling pathway were investigated in human A549 lung carcinoma cells treated with sodium dichromate. Sodium dichromate (100 microM, 0-6h) failed to activate nuclear factor kappa B (NF-kappaB) as determined by a lack of nuclear translocation of p65 but resulted in Jun N-terminal kinase activation as assessed by phospho-Jun N-terminal kinase Western blotting in a dose-dependent (>25 microM) and time-dependent (>1h) manner. In addition, c-Jun, a downstream target of Jun N-terminal kinase signalling was also activated with a similar dose- and time-dependency at the level of both protein expression and degree of phosphorylation. In contrast, sodium dichromate treatment had no effect on levels of phospho-p38. Immunoprecipitation demonstrated that apoptosis signal regulating kinase-1 (ASK-1), an upstream activator of Jun N-terminal kinase was dissociated from its inhibitory partner thioredoxin (Trx) in response to sodium dichromate (100 microM, 4h) treatment. This treatment was also associated with a transient (2h) increase in cytosolic levels of thioredoxin but no nuclear translocation of thioredoxin was observed. In conclusion, sodium dichromate had a stimulatory effect on the Jun N-terminal kinase signalling pathway in A549 cells, resulting in activation of downstream effector molecules. We hypothesise that dissociation of apoptosis signal regulating kinase-1 from thioredoxin may be at least partially responsible for Jun N-terminal kinase activation.

Apoptosis during spontaneous and prostaglandin F(2alpha)-induced luteal regression in the buffalo cow (Bubalus bubalis): involvement of mitogen-activated protein kinases.

The present study was conducted to evaluate whether the corpus luteum (CL) of the water buffalo (Bubalus bubalis) cow undergoes luteal regression by the process of apoptosis and to examine the involvement of mitogen-activated protein (MAP) kinases during prostaglandin (PG) F(2alpha)-induced luteolysis. Sections of CL from late in the estrous cycle, i.e., during spontaneous luteolysis, stained for 4',6'-diamidino-2-phenylindole revealed increased numbers of condensed nuclei, indicating cell death by apoptosis, which was confirmed further by the occurrence of pronounced oligonucleosome formation. For morphological and biochemical characterization during PGF(2alpha)-induced apoptosis, CL were collected at 0, 4, 12, and 18 h after injection of 750 micro g of Tiaprost, a synthetic analogue of PGF(2alpha), to midestrous buffalo cows. Serum progesterone concentrations fell within 4 h and decreased (P < 0.05) maximally by 18 h. Concomitant decreases (P < 0.05) in the levels of steroidogenic acute regulatory mRNA and protein were observed in CL during 12-18 h, with the more profound effect on mRNA levels. Quantitative analysis of the genomic DNA showed a >5-fold increase (P < 0.05) in the low molecular weight DNA fragments by 18 h postinjection. Immunoblot analysis of CL tissue lysates showed increased (P < 0.05) levels of phospho-Jun N-terminal kinase (JNK) 1 (4- to 14-fold during 4-18 h) and phospho-p38 (2- to 4-fold at 18 h). Immunohistochemical evaluation of CL sections revealed an increased nuclear localization of phospho-JNK after treatment. These findings demonstrate that the CL of the buffalo cow undergoes cell death by the process of apoptosis both during spontaneous and PGF(2alpha)-induced luteolysis and that MAP kinases are involved during PGF(2alpha)-mediated apoptosis in the CL.

Extranuclear sequestration of phospho-Jun N-terminal kinase and distorted villi produced by activated Rac1 in the intestinal epithelium of chimeric mice.

Previously, we used a genetic mosaic system to conduct an in vivo analysis of the effects of Rac1 activation on the developing intestinal epithelium ( Stappenbeck, T. S. and Gordon, J. I. (2000) Development 127, 2629-2642). Expression of a constitutively active human Rac1 (Rac1Leu61) in the 129/Sv-derived small intestinal epithelium of C57Bl/6-ROSA26<-->129/Sv chimeric mice led to precocious differentiation of some lineages with accompanying alterations in their apical actin. We have now explored the underlying mechanisms. Rac1Leu61 leads to accumulation of the 46 kDa form of phosphorylated Jun N-terminal kinase (p-Jnk) in the apical cytoplasm, but not in the nucleus of E18.5 proliferating and differentiating intestinal epithelial cells. The effect is cell-autonomous, selective for this mitogen-activated protein kinase family member, and accompanied by apical cytoplasmic accumulation of p21-activated kinase. c-Jun, a downstream nuclear target of p-Jnk, does not show evidence of enhanced phosphorylation, providing functional evidence for cytoplasmic sequestration of p-Jnk in Rac1Leu61-expressing epithelium. In adult chimeras, Rac1 activation augments cell proliferation in crypts of Lieberkühn, without a compensatory change in basal apoptosis and produces a dramatic, very unusual widening of villi. These results reveal a novel in vivo paradigm for Rac1 activation involving p-Jnk-mediated signaling at a distinctive extra-nuclear site, with associated alterations in the actin cytoskeleton. They also provide a new perspective about the determinants of small intestinal villus morphogenesis.