Transient Receptor Potential Melastatin 7 (TRPM7) Ion Channel Inhibitors: Preliminary SAR and Conformational Studies of Xenicane Diterpenoids from the Hawaiian Soft Coral Sarcothelia edmondsoni.

Waixenicin A, a xenicane diterpene from the octocoral Sarcothelia edmondsoni, is a selective, potent inhibitor of the TRPM7 ion channel. To study the structure-activity relationship (SAR) of waixenicin A, we isolated and assayed related diterpenes from S. edmondsoni. In addition to known waixenicins A (1) and B (2), we purified six xenicane diterpenes, 7S,8S-epoxywaixenicins A (3) and B (4), 12-deacetylwaixenicin A (5), waixenicin E (6), waixenicin F (7), and 20-acetoxyxeniafaraunol B (8). We elucidated the structures of 3-8 by NMR and MS analyses. Compounds 1, 2, 3, 4, and 6 inhibited TRPM7 activity in a cell-based assay, while 5, 7, and 8 were inactive. A preliminary SAR emerged showing that alterations to the nine-membered ring of 1 did not reduce activity, while the 12-acetoxy group, in combination with the dihydropyran, appears to be necessary for TRPM7 inhibition. The bioactive compounds are proposed to be latent electrophiles by formation of a conjugated oxocarbenium ion intermediate. Whole-cell patch-clamp experiments demonstrated that waixenicin A inhibition is irreversible, consistent with a covalent inhibitor, and showed nanomolar potency for waixenicin B (2). Conformational analysis (DFT) of 1, 3, 7, and 8 revealed insights into the conformation of waixenicin A and congeners and provided information regarding the stabilization of the proposed pharmacophore.

Spirovetivane- and Eudesmane-Type Sesquiterpenoids Isolated from the Culture Media of Two Cyanobacterial Strains.

As part of a study examining polar metabolites produced by cyanobacterial strains, we examined media extracts of a Calothrix sp. (strain R-3-1) and a Scytonema sp. (strain U-3-3). The cell mass of each was separated from the media, and HP20 resin was added for adsorption of secreted metabolites, a relatively unexplored area of cyanobacterial chemistry. HPLC-UV-LCMS-guided isolation led to the discovery of seven sesquiterpenoid compounds with five new, one known, and one previously isolated as the methyl ester. Through a complement of 1D and 2D NMR spectroscopic techniques, the planar structures and relative configurations of the seven compounds were elucidated. Spironostoic acid (1), 11,12-didehydrospironostoic acid (2), and 12-hydroxy-2-oxo-11-epi-hinesol (4) are spirovetivane-type compounds from R-3-1, while stigolone (5), 11R,12-dihydroxystigolone (6), and 11S,12-dihydroxystigolone (7) are three eudesmane-type compounds from U-3-3. Circular dichroism was utilized to decipher the absolute configurations of new compounds 1, 2, 4, 5, 6, and 7. Due to the structural variety observed among the spirovetivane- and eudesmane-type compounds in the literature and often a lack of clarity in how determinations were made, computational spectra and model compounds were used to support the interpretation of ECD and NMR spectra. A straightforward process to determine the configuration of these systems is presented.

The TRPM7 kinase limits receptor-induced calcium release by regulating heterotrimeric G-proteins.

The melastatin-related transient receptor potential member 7 (TRPM7) is a unique fusion protein with both ion channel function and enzymatic α-kinase activity. TRPM7 is essential for cellular systemic magnesium homeostasis and early embryogenesis; it promotes calcium transport during global brain ischemia and emerges as a key player in cancer growth. TRPM7 channels are negatively regulated through G-protein-coupled receptor-stimulation, either by reducing cellular cyclic adenosine monophosphate (cAMP) or depleting phosphatidylinositol bisphosphate (PIP2) levels in the plasma membrane. We here identify that heterologous overexpression of human TRPM7-K1648R mutant will lead to disruption of protease or purinergic receptor-induced calcium release. The disruption occurs at the level of Gq, which requires intact TRPM7 kinase phosphorylation activity for orderly downstream signal transduction to activate phospholipase (PLC)ß and cause calcium release. We propose that this mechanism may support limiting GPCR-mediated calcium signaling in times of insufficient cellular ATP supply.

Inhibiting Skp2 E3 Ligase Suppresses Bleomycin-Induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and no curative therapies. SCF-Skp2 E3 ligase is a target for cancer therapy, but there have been no reports about Skp2 as a target for IPF. Here we demonstrate that Skp2 is a promising therapeutic target for IPF. We examined whether disrupting Skp2 suppressed pulmonary fibrosis in a bleomycin (BLM)-induced mouse model and found that pulmonary fibrosis was significantly suppressed in Skp2-deficient mice compared with controls. The pulmonary accumulation of fibrotic markers such as collagen type 1 and fibronectin in BLM-infused mice was decreased in Skp2-deficient mice. Moreover, the number of bronchoalveolar lavage fluid cells accompanied with pulmonary fibrosis was significantly diminished. Levels of the Skp2 target p27 were significantly decreased by BLM-administration in wild-type mice, but recovered in Skp2-/- mice. In vimentin-positive mesenchymal fibroblasts, the decrease of p27-positive cells and increase of Ki67-positive cells by BLM-administration was suppressed by Skp2-deficency. As these results suggested that inhibiting Skp2 might be effective for BLM-induced pulmonary fibrosis, we next performed a treatment experiment using the Skp2 inhibitor SZL-P1-41. As expected, BLM-induced pulmonary fibrosis was significantly inhibited by SZL-P1-41. Moreover, p27 levels were increased by the SZL-P1-41 treatment, suggesting p27 may be an important Skp2 target for BLM-induced pulmonary fibrosis. Our study suggests that Skp2 is a potential molecular target for human pulmonary fibrosis including IPF.

Scalaradial Is a Potent Inhibitor of Transient Receptor Potential Melastatin 2 (TRPM2) Ion Channels.

TRPM2 is a Ca2+-permeable, nonselective cation channel that plays a role in oxidant-induced cell death, insulin secretion, and cytokine release. Few TRPM2 inhibitors have been reported, which hampers the validation of TRPM2 as a drug target. While screening our in-house marine-derived chemical library, we identified scalaradial and 12-deacetylscalaradial as the active components within an extract of an undescribed species of Cacospongia (class Demospongiae, family Thorectidae) that strongly inhibited TRPM2-mediated Ca2+ influx in TRPM2-overexpressing HEK293 cells. In whole-cell patch-clamp experiments, scalaradial (and similarly 12-deacetylscalaradial) inhibited TRPM2-mediated currents in a concentration- and time-dependent manner (∼20 min to full onset; IC50 210 nM). Scalaradial inhibited TRPM7 with less potency (IC50 760 nM) but failed to inhibit CRAC, TRPM4, and TRPV1 currents in whole-cell patch clamp experiments. Scalaradial's effect on TRPM2 channels was shown to be independent of its well-known ability to inhibit secreted phospholipase A2 (sPLA2) and its reported effects on extracellular signal-regulated kinases (ERK) and Akt pathways. In addition, scalaradial was shown to inhibit endogenous TRPM2 currents in a rat insulinoma cell line (IC50 330 nM). Based on its potency and emerging specificity profile, scalaradial is an important addition to the small number of known TRPM2 inhibitors.

Podocyte-specific NF-κB inhibition ameliorates proteinuria in adriamycin-induced nephropathy in mice.

BACKGROUND: Podocytes play a central role in the formation of the glomerular filtration barrier in the kidney, and their dysfunction has been shown to result in proteinuria. In the present study, we sought to determine the cell-autonomous role of NF-κB, a proinflammatory signaling, within podocytes in proteinuric kidney disease. METHODS: Podocyte-specific IκBΔN transgenic (Pod-IκBΔN) mice, in which NF-κB was inhibited specifically in podocytes, were generated by the Cre-loxP technology, and their phenotype was compared with control mice in adriamycin-induced nephropathy. RESULTS: Pod-IκBΔN mice were phenotypically normal and did not exhibit proteinuria at the physiological condition. By the intravenous administration of adriamycin, overt proteinuria appeared in Pod-IκBΔN mice, as well as in control mice. However, of interest, the amount of proteinuria was significantly lower in adriamycin-injected Pod-IκBΔN mice (373 ± 122 mg albumin/g creatinine), compared with adriamycin-injected control mice (992 ± 395 mg albumin/g creatinine). Expression of podocyte-selective slit diaphragm-associated proteins, such as nephrin and synaptopodin, was markedly decreased by adriamycin injection in control mice, whereas the reduction was attenuated in Pod-IκBΔN mice. Adriamycin-induced reduction in synaptopodin expression was also seen in cultured podocytes derived from control mice, but not in those from Pod-IκBΔN mice. CONCLUSIONS: Because nephrin and synaptopodin are essential for the maintenance of the slit diaphragm in podocytes, these results suggest that proteinuria in adriamycin-induced nephropathy is caused by the reduction in expression of these proteins. The results also suggest that the NF-κB signalling in podocytes cell-autonomously contributes to proteinuria through the regulation of these proteins.

TRPM7 kinase activity regulates murine mast cell degranulation.

KEY POINTS: The Mg(2+) and Ca(2+) conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function. Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not. As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release. Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function. Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca(2+) and extracellular Mg(2+) sensitivity of mast cell degranulation. ABSTRACT: Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7(+/∆K) ) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7(KR) ), are not hyperallergic and are resistant to systemic magnesium (Mg(2+) ) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7(+/+) ), TRPM7(+/∆K) and TRPM7(KR) mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg(2+) assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca(2+) ) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg(2+) caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca(2+) and affecting granular mobility and/or histamine contents.

Chk1 phosphorylates the tumour suppressor Mig-6, regulating the activation of EGF signalling.

The tumour suppressor gene product Mig-6 acts as an inhibitor of epidermal growth factor (EGF) signalling. However, its posttranslational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGF receptor (EGFR) activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1 depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts as a positive regulator of EGF signalling. This is a novel function of Chk1.

Roles of the Skp2/p27 axis in the progression of chronic nephropathy.

S-phase kinase-associated protein 2 (Skp2) is an F-box protein component of the Skp/Cullin/F-box-type E3 ubiquitin ligase that targets several cell cycle regulatory proteins for degradation through the ubiquitin-dependent pathway. Skp2-mediated degradation of p27, a cyclin-dependent kinase inhibitor, is involved in cell cycle regulation. Tubular epithelial cell proliferation is a characteristic feature of renal damage that is apparent in the early stages of nephropathy. The p27 level is associated with the progression of renal injury, and increased Skp2 expression in progressive nephropathy is implicated in decreases of p27 expression. In Skp2(-/-) mice, renal damage caused by unilateral ureteral obstruction (UUO) was ameliorated by p27 accumulation, mainly in tubular epithelial cells. However, the amelioration of UUO-induced renal injury in Skp2(-/-) mice was prevented by p27 deficiency in Skp2(-/-)/p27(-/-) mice. These results suggest that the Skp2-mediated reduction in p27 is a pathogenic activity that occurs during the progression of nephropathy. Here, we discuss the roles of the Skp2/p27 axis and/or related signaling pathways/components in the progression of chronic nephropathy.

Cannabigerolic Acid (CBGA) Inhibits the TRPM7 Ion Channel Through its Kinase Domain.

Cannabinoids are a major class of compounds produced by the plant Cannabis sativa. Previous work has demonstrated that the main cannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC) can have some beneficial effects on pain, inflammation, epilepsy, and chemotherapy-induced nausea and vomiting. While CBD and THC represent the two major plant cannabinoids, some hemp varieties with enzymatic deficiencies produce mainly cannabigerolic acid (CBGA). We recently reported that CBGA has a potent inhibitory effect on both Store-Operated Calcium Entry (SOCE) via inhibition of Calcium Release-Activated Calcium (CRAC) channels as well as currents carried by the channel-kinase TRPM7. Importantly, CBGA prevented kidney damage and suppressed mRNA expression of inflammatory cytokines through inhibition of these mechanisms in an acute nephropathic mouse model. In the present study, we investigate the most common major and minor cannabinoids to determine their potential efficacy on TRPM7 channel function. We find that approximately half of the tested cannabinoids suppress TRPM7 currents to some degree, with CBGA having the strongest inhibitory effect on TRPM7. We determined that the CBGA-mediated inhibition of TRPM7 requires a functional kinase domain, is sensitized by both intracellular Mg⋅ATP and free Mg2+ and reduced by increases in intracellular Ca2+. Finally, we demonstrate that CBGA inhibits native TRPM7 channels in a B lymphocyte cell line. In conclusion, we demonstrate that CBGA is the most potent cannabinoid in suppressing TRPM7 activity and possesses therapeutic potential for diseases in which TRPM7 is known to play an important role such as cancer, stroke, and kidney disease.

Decreased proteasomal activity causes age-related phenotypes and promotes the development of metabolic abnormalities.

The proteasome is a multicatalytic enzyme complex responsible for the degradation of both normal and damaged proteins. An age-related decline in proteasomal activity has been implicated in various age-related pathologies. The relevance of decreased proteasomal activity to aging and age-related diseases remains unclear, however, because suitable animal models are not available. In the present study, we established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity. Tg mice exhibited a shortened life span and developed age-related phenotypes. In Tg mice, polyubiquitinated and oxidized proteins accumulated, and the expression levels of cellular proteins such as Bcl-xL and RNase L were altered. When Tg mice were fed a high-fat diet, they developed more pronounced obesity and hepatic steatosis than did wild-type mice. Consistent with its role in lipid droplet formation, the expression of adipose differentiation-related protein (ADRP) was elevated in the livers of Tg mice. Of note, obesity and hepatic steatosis induced by a high-fat diet were more pronounced in aged than in young wild-type mice, and aged wild-type mice had elevated levels of ADRP, suggesting that the metabolic abnormalities present in Tg mice mimic those in aged mice. Our results provide the first in vivo evidence that decreased proteasomal chymotrypsin-like activity affects longevity and aggravates age-related metabolic disorders, such as obesity and hepatic steatosis.

CBGA ameliorates inflammation and fibrosis in nephropathy.

Cannabidiol (CBD) is thought to have multiple biological effects, including the ability to attenuate inflammatory processes. Cannabigerols (CBGA and its decarboxylated CBG molecule) have pharmacological profiles similar to CBD. The endocannabinoid system has recently emerged to contribute to kidney disease, however, the therapeutic properties of cannabinoids in kidney disease remain largely unknown. In this study, we determined whether CBD and CBGA can attenuate kidney damage in an acute kidney disease model induced by the chemotherapeutic cisplatin. In addition, we evaluated the anti-fibrosis effects of these cannabinoids in a chronic kidney disease model induced by unilateral ureteral obstruction (UUO). We find that CBGA, but not CBD, protects the kidney from cisplatin-induced nephrotoxicity. CBGA also strongly suppressed mRNA of inflammatory cytokines in cisplatin-induced nephropathy, whereas CBD treatment was only partially effective. Furthermore, both CBGA and CBD treatment significantly reduced apoptosis through inhibition of caspase-3 activity. In UUO kidneys, both CBGA and CBD strongly reduced renal fibrosis. Finally, we find that CBGA, but not CBD, has a potent inhibitory effect on the channel-kinase TRPM7. We conclude that CBGA and CBD possess reno-protective properties, with CBGA having a higher efficacy, likely due to its dual anti-inflammatory and anti-fibrotic effects paired with TRPM7 inhibition.

Up-regulation of Cks1 and Skp2 with TNFα/NF-κB signaling in chronic progressive nephropathy.

The cyclin-dependent kinase (CDK) inhibitor p27 level is associated with progression of renal damage. We previously reported that mRNA of Skp2, a component of Skp/Cullin/F-box (SCF)-ubiquitin ligase which targets to p27, was increased in unilateral ureteral obstructive kidneys in mice and that the nephritis was attenuated in Skp2-deficient mice. However, the details have not been fully clarified. Here, we found that not only Skp2 but also cdc kinase subunit 1 (Cks1), an essential cofactor for the SCF-Skp2 ubiquitin ligase in targeting p27, was increased in another chronic progressive model, anti-thymocyte serum (ATS) rat nephropathy. After induction of ATS nephropathy, Skp2(+) /Cks1(+) /Ki67(+) tubular epithelial cell numbers increased, and p27(+) tubular epithelial cells decreased transiently. Moreover, we found that TNFα was involved in expression of both Skp2 and Cks1 in NRK cell line as well as the in ATS nephropathy. Nuclear accumulations of NF-κB subunits RelB and p52 were increased in the tubular epithelial cells of the nephritic kidney. Both Skp2 and Cks1 were colocalized with RelB in these cells. These data suggest that both Skp2 and Cks1 are up-regulated by the TNFα-RelB/p52 pathway in the early stages of renal damage and are collaboratively involved in down-regulation of p27 in proliferative tubular dilation and the progression of chronic nephropathy.

Hydrogen gas and preservation of intestinal stem cells in mesenteric ischemia and reperfusion.

BACKGROUND: Patients with mesenteric ischemia frequently suffer from bowel necrosis even after revascularization. Hydrogen gas has showed promising effects for ischemia-reperfusion injury by reducing reactive oxygen species in various animal and clinical studies. We examined intestinal tissue injury by ischemia and reperfusion under continuous initiation of 3% hydrogen gas. AIM: To clarify the treatment effects and target cells of hydrogen gas for mesenteric ischemia. METHODS: Three rat groups underwent 60-min mesenteric artery occlusion (ischemia), 60-min reperfusion following 60-min occlusion (reperfusion), or ischemia-reperfusion with the same duration under continuous 3% hydrogen gas inhalation (hydrogen). The distal ileum was harvested. Immunofluorescence staining with caspase-3 and leucine-rich repeat-containing G-protein-coupled 5 (LGR5), a specific marker of intestinal stem cell, was conducted to evaluate the injury location and cell types protected by hydrogen. mRNA expressions of LGR5, olfactomedin 4 (OLFM4), hairy and enhancer of split 1, Jagged 2, and Neurogenic locus notch homolog protein 1 were measured by quantitative polymerase chain reaction. Tissue oxidative stress was analyzed with immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG). Systemic oxidative stress was evaluated by plasma 8-OHdG. RESULTS: Ischemia damaged the epithelial layer at the tip of the villi, whereas reperfusion induced extensive apoptosis of the cells at the crypt base, which were identified as intestinal stem cells with double immunofluorescence stain. Hydrogen mitigated such apoptosis at the crypt base, and the LGR5 expression of the tissues was higher in the hydrogen group than in the reperfusion group. OLFM4 was also relatively higher in the hydrogen group, whereas other measured RNAs were comparable between the groups. 8-OHdG concentration was high in the reperfusion group, which was reduced by hydrogen, particularly at the crypt base. Serum 8-OHdG concentrations were relatively higher in both reperfusion and hydrogen groups without significance. CONCLUSION: This study demonstrated that hydrogen gas inhalation preserves intestinal stem cells and mitigates oxidative stress caused by mesenteric ischemia and reperfusion.

Chitinase-like protein 3: A novel niche factor for mouse neural stem cells.

The concept of a perivascular niche has been proposed for neural stem cells (NSCs). This study examined endothelial colony-forming cell (ECFC)-secreted proteins as potential niche factors for NSCs. Intraventricle infusion with ECFC-secreted proteins increased the number of NSCs. ECFC-secreted proteins were more effective in promoting NSC self-renewal than marrow stromal cell (MSC)-secreted proteins. Differential proteomics analysis of MSC-secreted and ECFC-secreted proteins was performed, which revealed chitinase-like protein 3 (CHIL3; also called ECF-L or Ym1) as a candidate niche factor for NSCs. Experiments with recombinant CHIL3, small interfering RNA, and neutralizing antibodies demonstrated that CHIL3 stimulated NSC self-renewal with neurogenic propensity. CHIL3 was endogenously expressed in the neurogenic niche of the brain and retina as well as in the injured brain and retina. Transcriptome and phosphoproteome analyses revealed that CHIL3 activated various genes and proteins associated with NSC maintenance or neurogenesis. Thus, CHIL3 is a novel niche factor for NSCs.

Exclusive expression of proteasome subunit {beta}5t in the human thymic cortex.

The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three beta subunits, beta1, beta2, and beta5, of the 20S proteasome. Recently, a novel beta subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated beta5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human beta5t is expressed exclusively in the thymic cortex. Human beta5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human beta5t was incorporated into proteasomes with two other catalytically active beta subunits beta1i and beta2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.

Reduction of transforming growth factor-beta type II receptor is caused by the enhanced ubiquitin-dependent degradation in human renal cell carcinoma.

Although dysregulation of transforming growth factor-beta (TGF-beta) signaling is implicated in renal carcinogenesis, its precise mechanism is unknown in renal cell carcinoma (RCC). In our study, we investigated Smad-mediated TGF-beta signaling pathway and its regulatory mechanisms in surgical samples from patients with RCC. We found that immunoreactivity for nuclear phosphorylated Smad2 was significantly decreased in RCC compared to normal renal tissues, thereby TGF-beta signaling was suggested to be attenuated in RCC tissues. In accordance with the result, transcriptional downregulation of Smad4 and post-transcriptional downregulation of TGF-beta type II receptor (TbetaR-II) were frequently found in RCC tissues compared to normal renal tissues. Next, to clarify the reason why the protein level of TbetaR-II was decreased in RCC, we investigated the activities of degradation and ubiquitination of TbetaR-II. We found that both proteasome-mediated degradation and ubiquitination of TbetaR-II were markedly enhanced in RCC tissues. Moreover, we found that the level of Smad-ubiquitination regulatory factor 2 (Smurf2), the E3 ligase for TbetaR-II, was increased in RCC tissues of the patients with higher clinical stages compared to the normal tissues and was inversely correlated with the level of TbetaR-II. Our results suggest that the low TbetaR-II protein level is due to augmented ubiquitin-dependent degradation via Smurf2 and might be involved in the attenuation of TGF-beta signaling pathway in RCC.

Hepatocyte Growth Factor stimulated cell scattering requires ERK and Cdc42-dependent tight junction disassembly.

The mechanism by which Hepatocyte Growth Factor (HGF) induces tight junction disassembly prior to cell scattering is largely unknown. Here, we show that HGF stimulates rapid loss of the TJ assembly protein Par6 from the TJ in an Erk-dependent manner. Erk activation by HGF is found to mediate the interaction of Par6 with GTP-loaded Cdc42. The Cdc42 GTPase activating protein cdGAP is shown to interact with Pkcζ at baseline and prevent Par6-Cdc42 association. Erk, by phosphorylating cdGAP at threonine776, can inhibit the GAP activity, thereby increasing Par6-Cdc42 association and TJ disassembly. Our findings reveal a novel pathway for regulating HGF signaling to the Par proteins through Erk-cdGAP, resulting in TJ disassembly and cell scattering.

Excess iodide decreases transcription of NIS and VEGF genes in rat FRTL-5 thyroid cells.

Although it is well known that an excess of iodide suppresses thyroid function and blood flow in vivo, the underlying molecular mechanisms are not fully known. The functional effect of iodide occurs at multiple steps, which include inhibition of sodium/iodide symporter (NIS) expression, transient block of organification, and inhibition of hormonal release. The vascular effect likely involves suppression of the vascular endothelial growth factor (VEGF) gene. In this report, we show that excess iodide coordinately suppresses the expression of the NIS and VEGF genes in FRTL-5 thyroid cells. We also demonstrate that the mechanism of iodide suppression of NIS gene expression is transcriptional, which is synergized by the addition of thyroglobulin. Based on the findings of reporter gene assays and electrophoretic gel mobility shift analysis, we also report two novel DNA binding proteins that responded specifically to iodide and modulated NIS promoter activity. The results suggest that excess iodide affects thyroid vascular function in addition to iodide uptake. This study provides additional insights into the mechanism of action of excess iodide on thyroid function.

Decrease in tumor necrosis factor-alpha receptor-associated death domain results from ubiquitin-dependent degradation in obstructive renal injury in rats.

Increased expression levels of tumor necrosis factor-alpha (TNFalpha) is involved in tubulointerstitial cell proliferation and apoptosis in obstructive renal injury. Two TNFalpha receptors (TNFRs), TNFR1 and TNFR2, are known to exist. On TNFalpha binding, TNFR1 recruits TNFR-associated death domain (TRADD), an assembly platform to mediate TNFR1 signaling. We investigated postreceptor TRADD regulation in rat kidneys with unilateral ureteral obstruction (UUO). Whereas UUO was associated with increased expression levels of TNFalpha, TNFR1, TNFR2, and TRADD mRNAs, it resulted in the marked decrease of TRADD protein levels (which appeared at day 1 and persisted thereafter) and a slight decrease in TNFR1 protein levels at days 7 and 14. Both ubiquitination and degradation of TRADD were increased in UUO kidneys, degradation of TRADD was stimulated by TNFalpha in HK-2 cells, and TRADD degradation was suppressed by proteasome inhibitor. Inhibition of TNFalpha by soluble TNFR2, etanercept, reduced significantly, although transiently, tubular and interstitial cell proliferation, fibronectin expression, and apoptosis in UUO kidneys, and also suppressed TRADD degradation. These data suggest that the decrease in TRADD resulting from enhanced ubiquitin-dependent degradation is involved in obstructive renal injury. Since TRADD is not incorporated into TNFR2-mediated TNFalpha signaling, the persistent decrease in TRADD, associated with a mild decrease in TNFR1 levels, may function, at least in part, to divert TNFalpha signals toward a TNFR2-mediated pathway in UUO kidneys.