Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis.

BACKGROUND & AIMS: Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. METHODS: Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. RESULTS: Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1ß reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. CONCLUSIONS: After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation and cytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).

Dendritic cell-specific disruption of TGF-ß receptor II leads to altered regulatory T cell phenotype and spontaneous multiorgan autoimmunity.

In vitro data and transgenic mouse models suggest a role for TGF-ß signaling in dendritic cells (DCs) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHC class II (MHCII) and costimulatory molecules and increased expression of IDO, among others. To test whether a complete lack of TGF-ß signaling in DCs predisposes mice to spontaneous autoimmunity and to verify the mechanisms implicated previously in vitro, we generated conditional knockout (KO) mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 wk of age with multiorgan autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, costimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more proinflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated Foxp3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25(-)Foxp3(+) Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFN-γ and were not capable of directing Ag-specific Treg conversion unless in the presence of anti-IFN-γ blocking Ab. Adoptive transfer of induced Tregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGF-ß signaling in DCs is critical in the control of autoimmunity through both Treg-dependent and -independent mechanisms, but it does not affect MHCII and costimulatory molecule expression.

Cooperative role of NF-{kappa}B and poly(ADP-ribose) polymerase 1 (PARP-1) in the TNF-induced inhibition of PHEX expression in osteoblasts.

Reduced bone mass is a common complication in chronic inflammatory diseases, although the mechanisms are not completely understood. The PHEX gene encodes a zinc endopeptidase expressed in osteoblasts and contributes to bone mineralization. The aim of this study was to determine the molecular mechanism involved in TNF-mediated down-regulation of Phex gene transcription. We demonstrate down-regulation of the Phex gene in two models of colitis: naive T-cell transfer and in gnotobiotic IL-10(-/-) mice. In vitro, TNF decreased expression of Phex in UMR106 cells and did not require de novo synthesis of a transrepressor. Transfecting UMR-106 cells with a series of deletion constructs of the proximal Phex promoter identified a region located within -74 nucleotides containing NF-κB and AP-1 binding sites. After TNF treatment, the RelA/p50 NF-κB complex interacted with two cis-elements at positions -70/-66 and -29/-25 nucleotides in the proximal Phex promoter. Inhibition of NF-κB signaling increased the basal level of Phex transcription and abrogated the effects of TNF, whereas overexpression of RelA mimicked the effect of TNF. We identified poly(ADP-ribose) polymerase 1 (PARP-1) binding immediately upstream of the NF-κB sites and showed that TNF induced poly(ADP-ribosyl)ation of RelA when bound to the Phex promoter. TNF-mediated Phex down-regulation was completely abrogated in vitro by PARP-1 inhibitor and overexpression of poly(ADP-ribose) glucohydrolase (PARG) and in vivo in PARP-1(-/-) mice. Our results suggest that NF-κB signaling and PARP-1 enzymatic activity cooperatively contribute to the constitutive and inducible suppression of Phex. The described phenomenon likely contributes to the loss of bone mass density in chronic inflammatory diseases, such as inflammatory bowel disease.

Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis.

BACKGROUND & AIMS: Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS: We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS: Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS: The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

Colonic gene expression profile in NHE3-deficient mice: evidence for spontaneous distal colitis.

Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.