Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes

Background Sox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined. Results The active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level. Conclusions The increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this

Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases

Background. ClC-Ka and ClC-Kb, chloride channels participating in renal tubular Cl transport, require the coexpression of barttin to become functional. Mutations of the barttin gene lead to the Bartter's syndrome variant BSND, characterized by congenital deafness and severe renal salt wasting. Barttin bears a proline-tyrosine motif, a target structure for the ubiquitin ligase Nedd4-2, which mediates the clearance of channel proteins from the cell membrane. Nedd4-2 is, in turn, a target of the serum- and glucocorticoid-inducible kinase SGK1, which phosphorylates and, thus, inactivates the ubiquitin ligase. ClC-Ka also possesses a SGK1 consensus site in its sequence. We hypothesized that ClC-Ka/barttin is stimulated by SGK1, and down-regulated by Nedd4-2, an effect that may be reversed by SGK1 and its isoforms, SGK2 or SGK3.Methods. To test this hypothesis, ClC-Ka/barttin was heterologously expressed in Xenopus oocytes with or without the additional expression of Nedd4-2, SGK1, SGK2, SGK3, constitutively active (S422D)SGK1, or inactive (K127N)SGK1.Results. Expression of ClC-Ka/barttin induced a slightly inwardly rectifying current that was significantly decreased upon coexpression of Nedd4-2, but not the catalytically inactive mutant (C938S)Nedd4-2. The coexpression of (S422D)SGK1, SGK1, or SGK3, but not SGK2 or (K127N)SGK1 significantly stimulated the current. Moreover, (S422D)SGK1, SGK1, and SGK3 also phosphorylated Nedd4-2 and thereby inhibited Nedd4-2 binding to its target. The down-regulation of ClC-Ka/ barttin by Nedd4-2 was abolished by elimination of the PY motif in barttin.Conclusion. ClC-Ka/ barttin channels are regulated by SGK1 and SGK3, which may thus participate in the regulation of transport in kidney and inner ear

Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5

Contains fulltext : 118040.pdf (publisher's version ) (Open Access)Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5(682P/+)) and homozygous (Trpv5(682P/682P)) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and approximately 10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P) mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3) concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+) and Trpv5(682P/682P) mice consistent with a trafficking defect. In addition, Trpv5(682P/682P) mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K), consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria

Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+ exchanger NHE3 by glucocorticoids

SGK1 plays an important role in regulation of Na+/H+ exchanger (NHE) 3 in vivo. We show that SGK3 colocalizes with NHE3 in recycling endosomes. These studies identify SGK3 as the effector of the PI3K pathway that activates NHE3 and show that endosomal localization of SGK3 is essential for acute activation of NHE3

Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5.

Contains fulltext : 48619.pdf (publisher's version ) (Closed access)To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of GFP-TRPV5 in this cell line showed an intracellular granular distribution. Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on plastic supports was threefold higher than in non-transfected cells. Moreover, apical Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on permeable supports was eightfold higher than basolateral Ca2+ uptake, indicating that GFP-TRPV5 is expressed predominantly in the apical membrane. Patch-clamp analysis showed the presence of typical electrophysiological features of GFP-TRPV5, such as inwardly rectifying currents, inhibition by divalent cations and Ca2+-dependent inactivation. Moreover, the TRPV5 inhibitor ruthenium red completely inhibited Ca2+ uptake in GFP-TRPV5-MDCK cells, whereas Ca2+ uptake in non-transfected cells was not inhibited. The characterized GFP-TRPV5-MDCK cell line was used to assess the regulation of TRPV5. The protein kinase C activator phorbol 12-myristate 13-acetate and the cAMP-elevating compounds forskolin/3-isobutyl-1-methylxanthine, 8-Br-cAMP and PGE2 stimulated TRPV5 activity in GFP-TRPV5-MDCK cells by 121+/-7, 79+/-5, 55+/-4 and 61+/-7%, respectively. These compounds did not affect Ca2+ uptake in non-transfected cells. In conclusion, the GFP-TRPV5-MDCK cell line provides a model to specifically study the regulation of TRPV5 activity