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.

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.

TRPM7 contributes to progressive nephropathy.

TRPM7 belongs to the Transient Receptor Potential Melastatin family of ion channels and is a divalent cation-conducting ion channel fused with a functional kinase. TRPM7 plays a key role in a variety of diseases, including neuronal death in ischemia, cancer, cardiac atrial fibrillation, malaria invasion. TRPM7 is aberrantly over-expressed in lung, liver and heart fibrosis. It is also overexpressed after renal ischemia-reperfusion, an event that induces kidney injury and fibrosis. However, the role of TRPM7 in kidney fibrosis is unclear. Using the unilateral ureteral obstruction (UUO) mouse model, we examined whether TRPM7 contributes to progressive renal damage and fibrosis. We find that TRPM7 expression increases in UUO kidneys. Systemic application of NS8593, a known TRPM7 inhibitor, prevents kidney atrophy in UUO kidneys, retains tubular formation, and reduces TRPM7 expression to normal levels. Cell proliferation of both tubular epithelial cells and interstitial cells is reduced by NS8593 treatment in UUO kidneys, as are TGF-ß1/Smad signaling events. We conclude that TRPM7 is upregulated during inflammatory renal damage and propose that pharmacological intervention targeting TRPM7 may prove protective in progressive kidney fibrosis.

Hydrogen gas distribution in organs after inhalation: Real-time monitoring of tissue hydrogen concentration in rat.

Hydrogen has therapeutic and preventive effects against various diseases. Although animal and clinical studies have reported promising results, hydrogen distribution in organs after administration remains unclear. Herein, the sequential changes in hydrogen concentration in tissues over time were monitored using a highly sensitive glass microsensor and continuous inhalation of 3% hydrogen gas. The hydrogen concentration was measured in the brain, liver, kidney, mesentery fat and thigh muscle of rats. The maximum concentration, time to saturation, and other measurements representing the dynamics of distribution were obtained from the concentration curves, and the results obtained for different organs were compared. The time to saturation was significantly longer (20.2 vs 6.3-9.4 min. P = 0.004 in all cases) and increased more gradually in muscle than in the other organs. The maximum concentration was the highest in liver and the lowest in the kidney (29.0 ± 2.6 vs 18.0 ± 2.2 µmol/L; P = 0.03 in all cases). The concentration varied significantly depending on the organ (P = 0.03). These results provide the fundamentals for elucidating the mechanisms underlying the in vivo favourable effects of hydrogen gas in mammalian systems.

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.

Generation of kidney tubular organoids from human pluripotent stem cells.

Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3ß inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.

The coiled-coil domain of zebrafish TRPM7 regulates Mg·nucleotide sensitivity.

TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel's regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only.

Nestin protein is phosphorylated in adult neural stem/progenitor cells and not endothelial progenitor cells.

An intermediate filament protein, Nestin, is known as a neural stem/progenitor cell marker. It was shown to be required for the survival and self-renewal of neural stem cells according to the phenotypes of Nestin knockout mice. Nestin expression has also been reported in vascular endothelial cells, and we recently reported Nestin expression in proliferating endothelial progenitor cells, but not in mature endothelial cells. Using quantitative phosphoproteome analysis, we studied differences in phosphorylation levels between CNS Nestin in adult neural stem cells and vascular Nestin in adult bone-marrow-derived endothelial progenitor cells. We detected 495 phosphopeptides in the cell lysates of adult CNS stem/progenitor cells and identified 11 significant phosphorylated amino acid residues in the Nestin protein. In contrast, endothelial progenitor cells showed no significant phosphorylation of Nestin. We also measured neoplastic endothelial cells of the mouse brain and identified 13 phosphorylated amino acid residues in the Nestin protein. Among the 11 phosphorylated amino acids of adult CNS Nestin, five (S565, S570, S819, S883, and S886) were CNS Nestin-specific phosphorylation sites. Detection of the CNS-specific phosphorylation sites in Nestin, for example, by a phospho-specific Nestin antibody, may allow the expression of CNS Nestin to be distinguished from vascular Nestin.

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.

Identification of microRNAs that mediate thyroid cell growth induced by TSH.

TSH is a major regulator of thyroid cell growth and endocrine function. It is known that cAMP and phosphatidylinositol 3-kinase (PI3K) are responsible for mediating the action of TSH. Activation of these signals results in the induction of a series of transcription factors and cell cycle regulating proteins, which induce cell proliferation. In addition to such canonical transcriptional regulation, it was recently shown that microRNA (miRNA or miR) constitutes another key mechanism for the regulation of gene expression. However, whether TSH action is mediated by miRNA in the thyroid is unknown. In this study, we have performed miRNA microarray analysis and demonstrated that TSH significantly decreases expression of 47 miRNA in thyroid cells. Among these, we have shown, using their specific agonists, that overexpression of miR-16 and miR-195 suppressed cell cycle progression and DNA synthesis that was induced by TSH. In silico analysis predicted that Mapk8, Ccne1, and Cdc6, the expression of which was up-regulated by TSH, are potential target genes for these miRNA, and overexpression of miR-16 and miR-195 suppressed expression of these target genes. The decrease of miR-16 and miR-195 expression by TSH was reproduced by forskolin and N(6),2'-O-dibutyryladenosine cAMP and reversed by the protein kinase A inhibitor H89 and the PI3K inhibitor LY294002. These results suggest that TSH activates cAMP/protein kinase A and PI3K cascades to decrease miR-16 and miR-195, which induce Mapk8, Ccne1, and Cdc6 to activate cell proliferation.

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.

GSK3 regulates the expressions of human and mouse c-Myb via different mechanisms.

BACKGROUND: c-Myb is expressed at high levels in immature progenitors of all the hematopoietic lineages. It is associated with the regulation of proliferation, differentiation and survival of erythroid, myeloid and lymphoid cells, but decreases during the terminal differentiation to mature blood cells. The cellular level of c-Myb is controlled by not only transcriptional regulation but also ubiquitin-dependent proteolysis. We recently reported that mouse c-Myb protein is controlled by ubiquitin-dependent degradation by SCF-Fbw7 E3 ligase via glycogen synthase kinase 3 (GSK3)-mediated phosphorylation of Thr-572 in a Cdc4 phosphodegron (CPD)-dependent manner. However, this critical threonine residue is not conserved in human c-Myb. In this study, we investigated whether GSK3 is involved in the regulatory mechanism for human c-Myb expression. RESULTS: Human c-Myb was degraded by ubiquitin-dependent degradation via SCF-Fbw7. Human Fbw7 ubiquitylated not only human c-Myb but also mouse c-Myb, whereas mouse Fbw7 ubiquitylated mouse c-Myb but not human c-Myb. Human Fbw7 mutants with mutations of arginine residues important for recognition of the CPD still ubiquitylated human c-Myb. These data strongly suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner. Mutations of the putative GSK3 phosphorylation sites in human c-Myb did not affect the Fbw7-dependent ubiquitylation of human c-Myb. Neither chemical inhibitors nor a siRNA for GSK3ß affected the stability of human c-Myb. However, depletion of GSK3ß upregulated the transcription of human c-Myb, resulting in transcriptional suppression of γ-globin, one of the c-Myb target genes. CONCLUSIONS: The present observations suggest that human Fbw7 ubiquitylates human c-Myb in a CPD-independent manner, whereas mouse Fbw7 ubiquitylates human c-Myb in a CPD-dependent manner. Moreover, GSK3 negatively regulates the transcriptional expression of human c-Myb but does not promote Fbw7-dependent degradation of human c-Myb protein. Inactivation of GSK3 as well as mutations of Fbw7 may be causes of the enhanced c-Myb expression observed in leukemia cells. We conclude that expression levels of human and mouse c-Myb are regulated via different mechanisms.

The neural stem/progenitor cell marker nestin is expressed in proliferative endothelial cells, but not in mature vasculature.

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization.

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.

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.

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.

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.

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta.

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.

Expression of PDS/Pds, the Pendred syndrome gene, in endometrium.

Expression of the Pendred syndrome gene (PDS/Pds) is thought to be responsible for the iodide transport in the thyroid as well as the formation and function of the inner ear. Its mRNA is also expressed in the kidney and placenta. We report here that PDS and its encoded protein (pendrin) are also expressed in the endometrium. The RNA levels of rat PDS in the endometrium and kidney were much higher than those of the thyroid, opposite of the pattern of RNA expression in humans. In human endometrium, pendrin localization changed from the basal to apical surfaces of the epithelium during progression of the menstrual cycle. This suggests a possible role for pendrin in cationic ion transport required to maintain the physiological function of the endometrium. Since there is no evidence of endometrial abnormalities in patients with Pendred syndrome, it suggests the existence of a compensatory mechanisms for pendrin's function in the uterus.