Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells

doi:10.1016/j.bbrc.2011.09.117

Biochemical and Biophysical Research Communications

Volume 414, Issue 3, 28 October 2011, Pages 557-562

https://doi.org/10.1016/j.bbrc.2011.09.117 Get rights and content

Abstract

Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D₃ ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (−31 to −46 kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D₃ are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.

Highlights

► Both klotho mRNA spliceforms are regulated by 1,25-dihydroxyvitamin D (1,25D). ► Regulation occurs in cell lines from kidney proximal, distal and collecting tubules. ► Candidate vitamin D response elements (VDREs) found near mouse and human kl genes. ► Two human VDREs and one mouse VDRE are active in reporter gene assays. ► Klotho regulation by 1,25D may potentiate phosphaturic effect of FGF23.

Introduction

Alpha-klotho, hereafter referred to as klotho, is an anti-aging gene expressed predominately in kidney and brain choroid plexus [1], and at lower levels in several other tissues [2]. The full-length, membrane form of klotho (m-KL) consists of two similar domains (termed KL1 and KL2 [3]) with similarity to glycosyl hydrolases, a transmembrane domain and a short intracellular domain. m-KL acts as a coreceptor with fibroblast growth factor receptor-1 (FGFR1) [2] to bind fibroblast growth factor 23 (FGF23) and mediate phosphaturia to correct the hyperphosphatemia arising from 1,25-dihydroxyvitamin D (calcitriol, abbreviated 1,25D) [4] stimulation of intestinal calcium and phosphate absorption.

Proteolyzed klotho (p-KL) is generated by cleavage at the short transmembrane domain [5] and is shed into the circulation. The p-KL form has direct enzymatic effects in the proximal tubule to modify membrane proteins by removing sialic acid residues [6], thereby affecting TRPV5 and ROMK1 transporters, as well as insulin/IGFI and Wnt signaling [2].

An alternatively spliced klotho transcript encodes a 549-residue peptide in the human [3] and a 550-residue protein in mouse [7] (Fig. 1). This truncated klotho, if produced, contains a signal peptide without a transmembrane domain, and is herein designated as secreted klotho (s-KL).

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25D), is also proposed to have anti-aging effects [4]. 1,25D actions are mediated through the nuclear vitamin D receptor (VDR) binding directly to vitamin D responsive elements (VDREs) along with its RXR heterodimeric partner [4]. 1,25D-liganded VDR induces FGF23 in osteocytes to boost circulating FGF23 [8], which promotes phosphaturia to protect against hyperphosphatemia [4]. FGF23 also increases 1,25D degradation via induction of CYP24A1, and represses CYP27B1 to curb 1,25D biosynthesis [9]. The present study pursues the hypothesis that 1,25D regulates the expression of both membrane and soluble klotho forms in multiple kidney cell types to support FGF23 phosphaturic and vitamin D counter regulatory actions at the kidney, possibly exerting anti-aging effects.

Section snippets

Cell lines and real time PCR

Murine distal convoluted tubule (mpkDCT) cells were cultured as described [10]. All other cell lines, including human embryonic kidney (HEK) cells, murine inner medullary collecting duct (IMCD-3) cells, human proximal tubule (HK-2) cells, and simian kidney (COS-7) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured as recommended. Culture media, fetal bovine serum, and penicillin–streptomycin stocks were obtained from Gibco (Invitrogen Corp., Carlsbad

Klotho mRNA expression in response to 1,25D treatment

In order to detect the m-KL and the s-KL transcripts in IMCD-3 cells, two sets of primers were designed (Fig. 1B) for qrtPCR of total RNA prepared from cells treated with 10⁻⁸ M 1,25D for 24 h. Klotho transcripts were enhanced by 1,25D in IMCD-3 cells, with m-KL showing an average 2.9-fold increase and s-KL displaying an average of 4.5-fold as compared to vehicle control (Fig. 2A).

For human klotho, a single primer set was used for both the m-KL and s-KL transcripts (Fig. 1A). As shown in Fig. 2D,

Discussion

Potential regulation of klotho mRNA by 1,25D was inferred from studies reported by Tsujikawa et al. [21], who described the effect of pharmacologic and dietary vitamin D manipulations in mice on the expression of a 5.2 kb klotho mRNA species (representing m-KL [7]) as visualized by Northern blotting in whole kidney samples [21]. The 5.8 kb s-KL form was not evaluated by Tsujikawa et al. [21]. Herein we reinforce and extend these whole animal studies to show (a) significant regulation of both

Acknowledgments

This work was supported by National Institutes of Health grants to MRH and a SOLUR fellowship from the Arizona State University School of Life Sciences to REF.

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Vitamin D receptor controls expression of the anti-aging klotho gene in mouse and human renal cells

Abstract

Highlights

Introduction

Section snippets

Cell lines and real time PCR

Klotho mRNA expression in response to 1,25D treatment

Discussion

Acknowledgments

Biochem. Biophys. Res. Commun.

J. Steroid Biochem. Mol. Biol.

FEBS Lett.

J. Biol. Chem.

J. Steroid Biochem. Mol. Biol.

Kidney Int.

Biochim. Biophys. Acta

Mutation of the mouse klotho gene leads to a syndrome resembling ageing

Nature