Mycophenolic acid inhibits mesangial cell activation through p38 MAPK inhibition

Mesangial cell (MC) proliferation and extracellular matrix (ECM) accumulation are major pathologic features of chronic renal disease including chronic allograft nephropathy (CAN). Mycophenolic acid (MPA), a potent immunosuppressant, has emerged as a treatment to prevent CAN because it inhibits MC proliferation and ECM synthesis, but the mechanism involved has not been clarified. The present study examined relative role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) activation in inhibitory effect of MPA on MC activation. Growth arrested and synchronized primary rat MC (passages 7-11) were stimulated by PDGF 10 ng/ml in the presence and absence of clinically attainable dose of MPA (0-10 microM). Cell proliferation was assessed by [(3)H]thymidine incorporation, fibronectin and the activation of ERK and p38 MAPK by Western blot analysis, and total collagen by [(3)H]proline incorporation. PDGF increased cell proliferation by 4.6-fold, fibronectin secretion by 3.2-fold, total collagen synthesis by 1.8-fold, and the activation of ERK and 38 MAPK by 5.6-fold and 3.1-fold, respectively, compared to control. MPA, at doses inhibiting PDGF-induced MC proliferation and ECM synthesis, effectively blocked p38 MAPK activation but reduced ERK activation by 23% at maximal concentration tested (10 microM). Exogenous guanosine partially reversed the inhibition of MPA on p38 MAPK activation. Inhibitor of ERK or p38 MAPK suppressed PDGF-induced MC proliferation and ECM synthesis. In conclusion, MPA inhibits p38 MAPK activation leading to inhibiting proliferation and ECM synthesis in MC. Guanosine reduction is partially responsible for inhibitory effect of MPA on p38 MAPK activation in MC.     

The Protective Role of Interleukin-33 in Myocardial Ischemia and Reperfusion Is Associated with Decreased HMGB1 Expression and Up-Regulation of the P38 MAPK Signaling Pathway

Interleukin-33 (IL-33) plays a protective role in myocardial ischemia and reperfusion (I/R) injury, but the underlying mechanism was not fully elucidated. The present study was designed to investigate whether IL-33 protects against myocardial I/R injury by regulating both P38 mitogen-activated-protein kinase (P38 MAPK), which is involved in one of the downstream signaling pathways of IL-33, and high mobility group box protein 1 (HMGB1), a late pro-inflammatory cytokine. Myocardial I/R injury increased the level of IL-33 and its induced receptor (sST) in myocardial tissue. Compared with the I/R group, the IL-33 group had significantly lower cardiac injury (lower serum creatine kinase (CK), lactate dehydrogenase (LDH), and cTnI levels and myocardial infarct size), a suppressed inflammatory response in myocardial tissue (lower expression of HMGB1, IL-6, TNF-α and INF-γ) and less myocardial apoptosis (much higher Bcl-2/Bax ratio and lower cleaved caspase-3 expression). Moreover, IL-33 activated the P38 MAPK signaling pathway (up-regulating P-P38 expression) in myocardial tissue, and SB230580 partially attenuated the anti-inflammatory and anti-apoptosis effects of IL-33. These findings indicated that IL-33 protects against myocardial I/R injury by inhibiting inflammatory responses and myocardial apoptosis, which may be associated with the HMGB1 and P38 MAPK signaling pathways.     

Immune reponses in human mesangial cells regulated by emodin from Polygonum hypoleucum Ohwi

In the hope of identifying agents of therapeutic value in glomerulonephritis from Chinese herbs, we found that methanolic extracts of Polygonum hypoleucum Ohwi (P. hypoleucum Ohwi) inhibit human mesangial cells proliferation activated with interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) previously. This study was designed to identify bioactive components from P. hypoleucum Ohwi and elucidate their action mechanisms. We tested four anthraquinones emodin, emodin 1-O-beta-D-glucoside (49A), physcion (62A), and physcion 1-O-beta-D-glucoside (50A) purified from P. hypoleucum Ohwi for their effects on human mesangial cell proliferation and cytokines production in vitro. On a percentage basis, emodin had the highest suppressing activity on the human mesangial cells proliferation activated by IL-1beta and IL-6. The IC50 of emodin on human mesangial cells proliferation were 17.9+/-1.2 microM. In contrast to 49A, 50A, and 62A, emodin also decreased IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) production in human mesangial cells activated with IL-1beta and IL-6. The IC50 of emodin on IL-1beta, IL-6 and TNF-alpha production in activated human mesangial cells were 16.6+/-1.8 microM, 8.2+/-1.3 microM, and 9.5+/-1.6 microM, respectively. Moreover, IL-1beta and TNF-alpha mRNA expression in activated human mesangial cells was impaired by emodin. The intracellular free Ca2+ concentration ([Ca2+]i) in IL-1beta and IL-6 activated human mesangial cells was decreased by emodin. It is unlikely that cytotoxicity was involved because no cell deaths were observable. We hypothesize that the inhibitory mechanisms of emodin on activated human mesangial cells proliferation may be related to the impairments of gene expression and production of cytokines and [Ca2+]i in the cells.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.     

Prolonged renal allograft survival by donor interleukin-6 deficiency: association with decreased alloantibodies and increased intragraft T regulatory cells

Both humoral and cellular immune responses are involved in renal allograft rejection. Interleukin (IL)-6 is a regulatory cytokine for both B and Foxp3 (forkhead box P3)-expressing regulatory T (Treg) cells. This study was designed to investigate the impact of donor IL-6 production on renal allograft survival. Donor kidneys from IL-6 knockout (KO) vs. wild-type (WT) C57BL/6 mice (H-2(b)) were orthotopically transplanted to nephrotomized BALB/c mice (H-2(d)). Alloantibodies and Treg cells were examined by fluorescence-activated cell sorting analysis. Graft survival was determined by the time to graft failure. Here, we showed that a deficiency in IL-6 expression in donor kidneys significantly prolonged renal allograft survival compared with WT controls. IL-6 protein was upregulated in renal tubules and endothelium of renal allografts following rejection, which correlated with an increase in serum IL-6 compared with that in those receiving KO grafts or naive controls. The absence of graft-producing IL-6 or lower levels of serum IL-6 in the recipients receiving IL-6 KO allografts was associated with decreased circulating anti-graft alloantibodies and increased the percentage of intragraft CD4(+)CD25(+)Foxp3(+) Treg cells compared with those with WT allografts. In conclusion, the lack of graft-producing IL-6 significantly prolongs renal allograft survival, which is associated with reduced alloantibody production and/or increased intragraft Treg cell population, implying that targeting donor IL-6 may effectively prevent both humoral and cellular rejection of kidney transplants.     

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.     

Function of Rho-kinase in prostaglandin D2-induced interleukin-6 synthesis in osteoblasts

We have previously reported that prostaglandin D2 (PGD2) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGD2-stimulated IL-6 synthesis in MC3T3-E1 cells. PGD2 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGD2-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGD2-stimulated IL-6 synthesis. The PGD2-stimulated IL-6 synthesis was reduced by PD98059, a MEK inhibitor, and SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, but not SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). However, Y27632 and fasudil failed to affect the PGD2-induced phosphorylation of p44/p42 MAP kinase. On the other hand, Y27632 as well as fasudil markedly attenuated the PGD2-induced phosphorylation of p38 MAP kinase. In addition, PGD2 additively induced IL-6 synthesis in combination with endothelin-1 which induces IL-6 synthesis through p38 MAP kinase regulated by Rho-kinase. These results strongly suggest that Rho-kinase regulates PGD2-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Beta-endorphin regulation of MAPKs in cultured human articular chondrocytes: MAPK inhibitors prevent the increase of IL-1 beta protein levels during beta-endorphin stimulation

We investigated the effect of β-endorphin on the activities of mitogen-activated protein kinases in cultured human articular chondrocytes in order to elucidate its effect on cartilage. Monolayer cultures of chondrocytes obtained from patients undergoing total knee arthroplasty were treated with 60, 600, or 6000 ng/ml β-endorphin, or 100 ng/ml naltrexone combined with 600 ng/ml β-endorphin. The regulation of three major mitogen-activated protein kinases phosphorylation, ERKp44/p42, p38, and JNK, was determined by Western blotting. We also examined the influence of specific mitogen-activated protein kinase inhibitors on IL-1β protein levels during β-endorphin stimulation. The results demonstrate that β-endorphin, dependent on concentration and duration of stimulation, significantly affected the activation of the three mitogen-activated protein kinases in cultured human articular chondrocytes. Naltrexone in some cases significantly regulated the mitogen-activated protein kinases in different ways when added to β-endorphin 600 ng/ml. Furthermore, specific mitogen-activated protein kinase inhibitors hindered the increase of IL-1β during β-endorphin incubation. The effect of β-endorphin seen in this study is considered critical for the production of several mediators of cartilage damage in an arthritic joint.     

Defining the involvement of p38alpha MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase

Despite its lack of specificity, the inhibitor SB 203580 has been widely used to implicate p38 mitogen-activated protein kinase (MAPK) in the synthesis of many cytokines. Here we show unequivocally that the production of interleukin (IL)-1beta, IL-6, IL-10, and tumor necrosis factor alpha (TNFalpha) requires p38 MAPK activity by demonstrating that the inhibitory effects of SB 203580 were reversed by expression of an SB 203580-resistant form of p38alpha (SBR-p38alpha) that fails to bind to SB 203580. This strategy established the requirement for p38 activity for the lipopolysaccharide-stimulated production of IL-10, IL-1beta, and IL-6 by the monocytic cell WEHI 274 and the production of IL-6 and TNFalpha stimulated by ligation of the Fc-gamma receptor of the mast cell MC/9. Expression of SBR-p38alpha in primary macrophages abrogated the ability of SB 203580 to inhibit the lipopolysaccharide-stimulated production of TNFalpha but not of IL-10. Expression of SBR-p38alpha in primary T lymphocytes abrogated the ability of SB 203580 to inhibit the production of interferon-gamma induced by co-ligation of CD3 and CD28 but not the production of interferon-gamma or IL-10 induced by IL-12. These results suggest that the levels of p38 MAPK activity required for maximal cytokine production vary with different cytokines and stimuli.     

Rosiglitazone inhibits transforming growth factor-β1 mediated fibrogenesis in ADPKD cyst-lining epithelial cells

Background

Interstitial fibrosis plays an important role in progressive renal dysfunction in autosomal dominant polycystic kidney disease (ADPKD). In our previous studies, we confirmed that PPAR-γ agonist, rosiglitazone could protect renal function and prolong the survival of a slowly progressive ADPKD animal model by reducing renal fibrosis. However, the mechanism remains unknown.

Methods

Primary culture epithelial cells pretreated with TGF-β1 were incubated with rosiglitazone. Extracellular matrix proteins were detected using real-time PCR and Western blotting. MAPK and Smad2 phosphorylation were measured with western blot. ERK1/2 pathway and P38 pathway were inhibited with the specific inhibitors PD98059 and SB203580. The Smad2 pathway was blocked with the siRNA. To address whether PPAR-γ agonist-mediated inhibition of TGF-β1–induced collagen type I expression was mediated through a PPAR-γ dependent mechanism, genetic and pharmaceutical approaches were used to block the activity of endogenous PPARγ.

Results

TGF-β1-stimulated collagen type I and fibronectin expression of ADPKD cyst-lining epithelia were inhibited by rosiglitazone in a dosage-dependent manner. Smad2, ERK1/2 and P38 pathways were activated in response to TGF-β1; however, TGF-β1 had little effect on JNK pathway. Rosiglitazone suppressed TGF-β1 induced Smad2 activation, while ERK1/2 and P38MAPK signals remained unaffected. Rosiglitazone could also attenuate TGF-β1-stimulated collagen type I and fibronectin expression in primary renal tubular epithelial cells, but had no effect on TGF-β1–induced activation of Smad2, ERK1/2 and P38 pathways. There was no crosstalk between the Smad2 and MAPK pathways in ADPKD cyst-lining epithelial cells. These inhibitory effects of rosiglitazone were reversed by the PPARγ specific antagonist GW9662 and PPARγ siRNA.

Conclusion

ADPKD cyst-lining epithelial cells participate in TGF-β1 mediated fibrogenesis. Rosiglitazone could suppress TGF-β1–induced collagen type I and fibronectin expression in ADPKD cyst-lining epithelia through modulation of the Smad2 pathway. Our study may provide therapeutic basis for clinical applications of rosiglitazone in retarding the progression of ADPKD.     

G protein-coupled receptor kinase 6 acts as a critical regulator of cytokine-induced hyperalgesia by promoting phosphatidylinositol 3-kinase and inhibiting p38 signaling

The molecular mechanisms determining magnitude and duration of inflammatory pain are still unclear. We assessed the contribution of G protein–coupled receptor kinase (GRK)-6 to inflammatory hyperalgesia in mice. We showed that GRK6 is a critical regulator of severity and duration of cytokine-induced hyperalgesia. In GRK6^−/− mice, a significantly lower dose (100 times lower) of intraplantar interleukin (IL)-1β was sufficient to induce hyperalgesia compared with wild-type (WT) mice. In addition, IL-1β hyperalgesia lasted much longer in GRK6^−/− mice than in WT mice (8 d in GRK6^−/− versus 6 h in WT mice). Tumor necrosis factor (TNF)-α–induced hyperalgesia was also enhanced and prolonged in GRK6^−/− mice. In vitro, IL-1β–induced p38 phosphorylation in GRK6^−/− dorsal root ganglion (DRG) neurons was increased compared with WT neurons. In contrast, IL-1β only induced activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway in WT neurons, but not in GRK6^−/− neurons. In vivo, p38 inhibition attenuated IL-1β– and TNF-α–induced hyperalgesia in both genotypes. Notably, however, whereas PI 3-kinase inhibition enhanced and prolonged hyperalgesia in WT mice, it did not have any effect in GRK6-deficient mice. The capacity of GRK6 to regulate pain responses was also apparent in carrageenan-induced hyperalgesia, since thermal and mechanical hypersensitivity was significantly prolonged in GRK6^−/− mice. Finally, GRK6 expression was reduced in DRGs of mice with chronic neuropathic or inflammatory pain. Collectively, these findings underline the potential role of GRK6 in pathological pain. We propose the novel concept that GRK6 acts as a kinase that constrains neuronal responsiveness to IL-1β and TNF-α and cytokine-induced hyperalgesia via biased cytokine-induced p38 and PI 3-kinase/Akt activation.     

The protective effects of Achyranthes bidentata root extract on the antimycin A induced damage of osteoblastic MC3T3-E1 cells

Achyranthes bidentata (A. bidentata) Blume is a medicinal herb with the property of strengthening bones and muscles and ensuring proper downward flow of blood in terms of the therapeutic theory of traditional medicine. In the present study, the effect of A. bidentata root extract (AE) on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. AE caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, and mineralization in the cells (P < 0.05). AE also decreased the production of TNF-α, IL-6, and RANKL induced by antimycin A, mitochondrial electron transport inhibitor. Exposure of MC3T3-E1 cells to antimycin A caused significant reduction of cell viability and mineralization. However, pretreatment with AE prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, ATP loss, ROS release, and nitrotyrosine increase, suggesting that AE may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, AE increased the phosphorylation of cAMP-response element-binding protein inhibited by antimycin A. Our study demonstrates that A. bidentata could significantly prevent osteoblast damage in aged patients.     

Study on the synthesis of complement components by human renal mesangial cells in culture. Assessment of the effect of cytokines

Renal mesangial cell (MC) cultures are easily established and widely used. MC produce some complement (C) regulatory proteins. We studied whether MC synthesize C components (C3, C5, C8). MC cultures were established from normal portions of cortices of nephrectomies for renal cancer. After growing to near-confluence in RPMI/17% FBS and resting for 24 h in RPMI/0.5% FBS, MC were stimulated up to 72 h with IL-1 or IL-6 (10, 100, 1000 U/ml). Neither C5 nor C8 were detected by ELISA. While C3 was present in supernatant under basal conditions (15.5–107.6 ng/10⁶ cells/24h) in different MC lines. IL-1 up-regulated the synthesis by 2.4–4.5 folds, whereas IL-6 did not show any effect. C3 synthetic rate was 1,76 ng/h/10⁶ cells under IL-1 stimulation versus basal rate of 0,37 ng/h/10⁶ cells. MC production of C3, especially induced by IL-1 may have pathogenetic relevance in glomerulonephritis.     

Interleukin-6 induces Gr-1+CD11b+ myeloid cells to suppress CD8+ T cell-mediated liver injury in mice

Background

Agonist antibodies against CD137 (4–1BB) on T lymphocytes are used to increase host anti-tumor immunity, but often leading to severe liver injury in treated mice or in patients during clinical trials. Interleukin-6 (IL-6) has been reported to protect hepatocyte death, but the role of IL-6 in protecting chronic T cell-induced liver diseases is not clearly defined due to lack of relevant animal models. We aimed to define the role of IL-6 in CD8+ T cell-mediated liver injury induced by a CD137 agonistic mAb (clone 2A) in mice.

Methods/Principal Findings

We expressed IL-6 in the liver by hydrodynamic gene delivery in mice treated with 2A or control mAb and studied how IL-6 treatment affected host immunity and T cell-mediated liver injury. We found that ectopic IL-6 expression in the liver elevated intrahepatic leukocyte infiltration but prevented CD8+ T cell-mediated liver injury. In IL-6 treated mice, CD8+ T cells proliferation and IFN-γ expression were inhibited in the liver. We discovered that IL-6 increased accumulation of Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the liver and spleen. These MDSCs had the ability to inhibit T cells proliferation and activation. Finally, we showed that the MDSCs were sufficient and essential for IL-6-mediated protection of anti-CD137 mAb-induced liver injury.

Conclusions/Significance

We concluded that IL-6 induced Gr-1+CD11b+ MDSCs in the liver to inhibit T cell-mediated liver injury. The findings have defined a novel mechanism of IL-6 in protecting liver from CD8+ T cell-mediated injury.     

IL-18 mediates proapoptotic signaling in renal tubular cells through a Fas ligand-dependent mechanism

Renal tubular cell apoptosis is a significant component of obstruction-induced renal injury, and it results in a progressive loss in renal parenchymal mass during renal obstruction. Although IL-18 is an important mediator of inflammatory renal disease and renal fibrosis, its role in obstruction-induced renal tubular cell apoptosis remains unclear. To study this, male C57BL6 wild-type mice and C57BL6 mice transgenic for human IL-18-binding protein (IL-18BP Tg) were subjected to renal obstruction vs. sham operation. The kidneys were harvested after 1 or 2 wk and analyzed for IL-18 production, apoptosis, caspase activity, and Fas/Fas Ligand (FasL) expression. HK-2 cells were similarly analyzed for apoptosis and proapoptotic signaling following 3 days of direct exposure to IL-18 vs. control media. Renal obstruction induced a significant increase in IL-18 production, renal tubular cell apoptosis, caspase activation, and FasL expression. IL-18 neutralization, on the other hand, significantly reduced obstruction-induced apoptosis, caspase-8 and caspase-3 activity, and FasL expression. In vitro experiments similarly demonstrate that IL-18 stimulation induces apoptosis, FasL expression, and increases active caspase-8 and caspase-3 expression in a dose-dependent fashion. siRNA knockdown of FasL gene expression, however, significantly reduced IL-18-induced apoptosis. This study reveals that IL-18 is a significant mediator of obstruction-induced tubular cell apoptosis, and it demonstrates that IL-18 stimulates proapoptotic signaling through a FasL-dependent mechanism.     

TBX3, a downstream target of TGF-β1, inhibits mesangial cell apoptosis

Chronic kidney disease (CKD) is an increasingly common condition characterized by progressive loss of functional nephrons leading to renal failure. TGF-β1-induced mesangial cell (MC) phenotype alterations have been linked to the genesis of CKD. Here we show that TGF-β1 regulates TBX3 gene expression in MC. This gene encodes for two main isoforms, TBX3.1 and TBX3+2α. TBX3.1 has been implicated in cell immortalization, proliferation and apoptosis by inhibiting p14^ARF-Mdm2-p53 pathway, while TBX3+2α role has not been defined. We demonstrated that TBX3 overexpression abrogated MC apoptosis induced by serum deprivation. Moreover, we observed an enhancement in TBX3 protein expression both in glomerular and tubular regions in the model of 5/6 nephrectomy, temporally related to increased expression of TGF-β1, type IV collagen and fibronectin. Our results indicate that TBX3 acts as an anti-apoptotic factor in MC in vitro and may be involved in the mechanism by which TGF-β1 induces glomerulosclerosis and tubular fibrosis during the progression of nephropathies.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Sublytic C5b‐9 induces proliferation of glomerular mesangial cells via ERK5/MZF1/RGC‐32 axis activated by FBXO28‐TRAF6 complex

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b‐9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy‐1 nephritis (Thy‐1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC‐32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal‐regulated kinase 5 (phosphorylated ERK5, p‐ERK5), F‐box only protein 28 (FBXO28) and TNF receptor‐associated factor 6 (TRAF6) were all markedly up‐regulated both in the renal tissues of rats with Thy‐1N (in vivo) and in the GMCs upon sublytic C5b‐9 stimulation (in vitro). Further in vitro experiments revealed that up‐regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63‐ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1‐dependent RGC‐32 up‐regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1‐binding element within RGC‐32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC‐32 genes respectively markedly reduced GMC proliferation and ECM production in Thy‐1N rats. Together, these findings indicate that sublytic C5b‐9 induces GMC proliferative changes in rat Thy‐1N through ERK5/MZF1/RGC‐32 axis activated by the FBXO28‐TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.     

Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-β activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1β-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1β induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1β-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1β-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.