Klotho was discovered as an antiaging gene, and a-Klotho (Klotho) is expressed in multiple tissues with a broad set of biologic functions. Membrane-bound Klotho binds fibroblast growth factor 23 (FGF23), but a soluble form of Klotho is also produced by alternative splicing or cleavage of the extracellular domain of the membrane-bound protein. The relative organ-specific contributions to the levels and effects of circulating Klotho remain unknown. We explored these issues by generating a novel mouse strain with Klotho deleted throughout the nephron (Six2-KL 2/2 ). Klotho shedding from Six2-KL

BackgroundIn vivo models of uremia are important tools to study numerous aspects of acute and chronic kidney disease. Mouse models are pivotal because most genetically engineered animal models are mice, which allow dissecting the impact of selected target genes in renal failure. Adenine-based protocols to induce renal failure are available in rats, but have not been adapted in mice due to their reluctance to consume adenine. In the current paper we developed a novel method for induction of renal failure through dietary delivery of adenine mixed in a casein-based diet.ResultsAfter an induction phase, a stable model of renal impairment was obtained (target urea range 80–100 mg/dL), mimicking several aspects of chronic kidney disease - mineral and bone disorder including secondary hyperparathyroidism, bone abnormalities and pathological elevation of FGF23. No deaths occurred and the level of uremia was adaptable through adjustments of the adenine content, providing significant advantages compared to existing models. In an 8-week proof-of-concept study, renal histology showed mainly a tubulointerstitial damage with infiltrating leukocytes, interstitial edema and widening of the Bownman's space. Fibrosis was present in most animals as defined by histology and gene expression changes of fibrosis markers. Parathyroid cell proliferation was markedly increased but without signs of glandular hypertrophy. Skeletal histology showed increased trabecular bone and bone marrow adiposity whereas bone biomarkers (CTX and PINP) suggested higher bone formation, but surprisingly, lower bone resorption and perturbations in mineral metabolism.ConclusionsWe present a novel, non-surgical method for induction of renal failure in mice. This is an important complement to existing uremic models for pathophysiological studies in acute and chronic kidney disease, especially in terms of tubulointerstitial lesions.

Estrogen receptor B (ERB) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERB expression is reduced in colorectal cancer. Our hypothesis is that ERB inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERB in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERB expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERB resulted in inhibition of proliferation and G 1 phase cell cycle arrest and this effect was dependent on a functional DNA-binding region. c-Myc is overexpressed in an overwhelming majority of colorectal tumors. By Western blot and real-time PCR, we found c-Myc to be down-regulated in the ERB-expressing cells. Furthermore, the c-Myc target gene p21 (Waf1/Cip1) was induced and Cdc25A was reduced by ERB at the transcriptional level. The second cdk2-inhibitor, p27 Kip1 , was induced by ERB, but this regulation occurred at the posttranscriptional level, probably through ERB-mediated repression of the F-box protein p45 Skp2 . Expression of the ERB-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells F ERB transplanted into severe combined immunodeficient/beige mice; after three weeks of ERB-expression, a 70% reduction of tumor volume was seen. Our results show that ERB inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERB-mediated inhibition of cell-cycle pathways. Furthermore, this ERB-mediated cell cycle repression is dependent on functional ERE binding. [Cancer Res 2009;69(15):6100-6]

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERA and ERB. ERA mediates the proliferative effect of estrogen in breast cancer cells, whereas ERB seems to be antiproliferative. We engineered ERA-positive T47D breast cancer cells to express ERB in a Tet-Off-regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERB. The presence of ERB resulted in a reduction in tumor growth. Comparison of the ERB-expressing and non-ERB-expressing tumors revealed that the expression of ERB caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor B (PDGFB). In cell culture, with the Tet-Off-regulated ERB-expressing cells, expression of ERB decreased expression of VEGF and PDGFB mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFB proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFB promoter constructs revealed a repressive effect of ERB at the promoter level of these genes. Taken together, these data show that introduction of ERB into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)

Klotho acts as a co-receptor for and dictates tissue specificity of circulating FGF23. FGF23 inhibits PTH secretion, and reduced Klotho abundance is considered a pathogenic factor in renal secondary hyperparathyroidism. To dissect the role of parathyroid gland resident Klotho in health and disease, we generated mice with a parathyroid-specific Klotho deletion (PTH-KL−/−). PTH-KL−/− mice had a normal gross phenotype and survival; normal serum PTH and calcium; unaltered expression of the PTH gene in parathyroid tissue; and preserved PTH response and sensitivity to acute changes in serum calcium. Their PTH response to intravenous FGF23 delivery or renal failure did not differ compared to their wild-type littermates despite disrupted FGF23-induced activation of the MAPK/ERK pathway. Importantly, calcineurin-NFAT signaling, defined by increased MCIP1 level and nuclear localization of NFATC2, was constitutively activated in PTH-KL−/− mice. Treatment with the calcineurin-inhibitor cyclosporine A abolished FGF23-mediated PTH suppression in PTH-KL−/− mice whereas wild-type mice remained responsive. Similar results were observed in thyro-parathyroid explants ex vivo. Collectively, we present genetic and functional evidence for a novel, Klotho-independent, calcineurin-mediated FGF23 signaling pathway in parathyroid glands that mediates suppression of PTH. The presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action.

Renal Klotho controls mineral metabolism by directly modulating tubular reabsorption of phosphate and calcium and by acting as a co-receptor for the phosphaturic and vitamin D-regulating hormone fibroblast growth factor-23 (FGF23). Klotho null mice have a markedly abnormal phenotype. We sought to determine effects of renal-specific and partial deletion of Klotho to facilitate investigation of its roles in health and disease. We generated a mouse model with partial deletion of Klotho in distal tubular segments (Ksp-KL 2/2 ).In contrast to Klotho null mice, Ksp-KL 2/2 mice were fertile, had a normal gross phenotype, and did not have vascular or tubular calcification on renal histology. However, Ksp-KL 2/2 mice were hyperphosphatemic with elevated FGF23 levels and abundant expression of the sodium-phosphate cotransporter Npt2a at the brush border membrane. Serum calcium and 1,25-dihydroxyvitamin D 3 levels were normal but parathyroid hormone levels were decreased. TRPV5 protein was reduced with a parallel mild increase in urinary calcium excretion. Renal expression of vitamin D regulatory enzymes and vitamin D receptor was higher in Ksp-KL 2/2 mice than controls, suggesting increased turnover of vitamin D metabolites and a functional increase in vitamin D signaling. There was a threshold effect of residual renal Klotho expression on FGF23: deletion of .70% of Klotho resulted in FGF23 levels 30-250 times higher than in wild-type mice. A subgroup of Ksp-KL 2/2 mice with normal phosphate levels had elevated FGF23, suggesting a Klothoderived renal-bone feedback loop. Taken together, renal FGF23-Klotho signaling, which is disrupted in CKD, is essential for homeostatic control of mineral metabolism.

Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (Sm22-KL−/−). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between Sm22-KL−/− and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine ex vivo model of endothelial function, respectively. FGF23 treatment (0.125–2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen ex vivo. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.

IntroductionThe inhibition of estrogen receptor (ER) α action with the ER antagonist tamoxifen is an established treatment in the majority of breast cancers. De novo or acquired resistance to this therapy is common. Expression of ERβ in breast tumors has been implicated as an indicator of tamoxifen sensitivity. The mechanisms behind this observation remain largely uncharacterized. In the present study, we investigated whether ERβ can modulate pathways implicated in endocrine resistance development.MethodsT47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERβ were used as a model system. Expression levels and activity of known regulators of endocrine resistance were analyzed by performing quantitative polymerase chain reaction assays, Western blot analysis and immunostaining, and sensitivity to tamoxifen was investigated by using a cell proliferation kit.ResultsExpression of ERβ in ERα-positive T47-D and MCF-7 human breast cancer cells resulted in a decrease in Akt signaling. The active form of an upstream regulator of Akt, proto-oncogene c-ErbB-2/receptor tyrosine kinase erbB-3 (HER2/HER3) receptor dimer, was also downregulated by ERβ. Furthermore, ERβ increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERβ expression increased the sensitivity of these breast cancer cells to tamoxifen.ConclusionsOur results suggest a link between expression of ERβ and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERβ presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for also using ERβ as a target in selected groups of breast cancer.