Growth Differentiation Factor 7 promotes multiple-lineage differentiation in tenogenic cultures of mesenchymal stem cells.

The repair of the tendon-bone interface, which is composed of tendon, fibrocartilage, and bony attachment, remains a clinical challenge. The application of mesenchymal stem cells (MSCs), collagen-rich extracellular matrix (ECMs), as well as growth factors, has the potential to regenerate this special multiple-tissue structure through the so-called biological augmentation. We present here an in vitro tendon regeneration model with C3H10T1/2 cells cultured on Collagen I matrix and evaluated the lineage determination effects of Growth Differentiation Factor 7 (GDF-7). We found that besides tenogenic effect, GDF-7 also stimulates the expression of osteoblastic as well as adipocytic genes. Our results indicate that GDF-7 might be a promising growth factor for regeneration of the tendon-bone interface due to its multiple-lineage stimulating effects. However, the side effect on adipogenic differentiation should be of concern, as it is a known risk factor for repair failures.

The kidney is the principal organ mediating klotho effects.

Klotho was discovered as an antiaging gene, and α-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(-/-)). Klotho shedding from Six2-KL(-/-) kidney explants was undetectable and the serum Klotho level was reduced by approximately 80% in Six2-KL(-/-) mice compared with wild-type littermates. Six2-KL(-/-) mice exhibited severe growth retardation, kyphosis, and premature death, closely resembling the phenotype of systemic Klotho knockout mice. Notable biochemical changes included hyperphosphatemia, hypercalcemia, hyperaldosteronism, and elevated levels of 1,25-dihydroxyvitamin D and Fgf23, consistent with disrupted renal Fgf23 signaling. Kidney histology demonstrated interstitial fibrosis and nephrocalcinosis in addition to absent dimorphic tubules. A direct comparative analysis between Six2-KL(-/-) and systemic Klotho knockout mice supports extensive, yet indistinguishable, extrarenal organ manifestations. Thus, our data reveal the kidney as the principal contributor of circulating Klotho and Klotho-induced antiaging traits.

Parathyroid-specific deletion of Klotho unravels a novel calcineurin-dependent FGF23 signaling pathway that regulates PTH secretion.

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.

A novel model of adenine-induced tubulointerstitial nephropathy in mice.

BACKGROUND: In 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. RESULTS: After 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. CONCLUSIONS: We 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.

Arterial klotho expression and FGF23 effects on vascular calcification and function.

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.

Targeted deletion of Klotho in kidney distal tubule disrupts mineral metabolism.

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(-/-)). In contrast to Klotho null mice, Ksp-KL(-/-) mice were fertile, had a normal gross phenotype, and did not have vascular or tubular calcification on renal histology. However, Ksp-KL(-/-) 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(-/-) 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(-/-) mice with normal phosphate levels had elevated FGF23, suggesting a Klotho-derived renal-bone feedback loop. Taken together, renal FGF23-Klotho signaling, which is disrupted in CKD, is essential for homeostatic control of mineral metabolism.

Estrogen receptor ß represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity.

INTRODUCTION: The 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 ERß 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 ERß can modulate pathways implicated in endocrine resistance development. METHODS: T47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERß 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. RESULTS: Expression of ERß 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 ERß. Furthermore, ERß increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERß expression increased the sensitivity of these breast cancer cells to tamoxifen. CONCLUSIONS: Our results suggest a link between expression of ERß 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 ERß 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 ERß as a target in selected groups of breast cancer.

Estrogen receptor beta decreases survival of p53-defective cancer cells after DNA damage by impairing G2/M checkpoint signaling.

Estrogen receptor beta (ERß) inhibits proliferation in different cellular systems by regulating components of the cell cycle machinery. Eukaryotic cells respond to DNA damage by arresting in G1, S, or G2 phases of the cell cycle to initiate DNA repair. Most tumor cells due to disruptions in the p53-dependent G1 pathway are dependent on S-phase and G2/M checkpoints to maintain genomic integrity in response to DNA damage. We report that induction of ERß expression causes abrogation of the S-phase, and the Chk1/Cdc25C-mediated G2/M checkpoints after cisplatin and doxorubicin exposure in p53-defective breast cancer cells but not in p53 wild-type mammary cells. This impairment of DNA damage response that involves BRCA1 downregulation and caspase-2 activation results in mitotic catastrophe and decreased cancer cell survival. These results indicate that in cancers where p53 is defective, assessment of the presence of ERß may be of predictive value for the successful response to chemotherapy.

Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells.

Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.

Tumor repressive functions of estrogen receptor beta in SW480 colon cancer cells.

Estrogen receptor beta (ERbeta) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERbeta expression is reduced in colorectal cancer. Our hypothesis is that ERbeta inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERbeta in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERbeta expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERbeta 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 ERbeta-expressing cells. Furthermore, the c-Myc target gene p21((Waf1/Cip1)) was induced and Cdc25A was reduced by ERbeta at the transcriptional level. The second cdk2-inhibitor, p27(Kip1), was induced by ERbeta, but this regulation occurred at the posttranscriptional level, probably through ERbeta-mediated repression of the F-box protein p45(Skp2). Expression of the ERbeta-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells +/- ERbeta transplanted into severe combined immunodeficient/beige mice; after three weeks of ERbeta-expression, a 70% reduction of tumor volume was seen. Our results show that ERbeta inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERbeta-mediated inhibition of cell-cycle pathways. Furthermore, this ERbeta-mediated cell cycle repression is dependent on functional ERE binding.

Different roles of estrogen receptors alpha and beta in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line.

Two estrogen receptors (ERalpha and ERbeta) are found throughout the mammary gland. Evidence indicates that, while ERalpha transduces proliferation signals, ERbeta opposes this effect and is necessary for epithelial differentiation. Using mouse mammary epithelial cells, we have previously shown that activation of ERbeta opposes ERalpha-induced proliferation and increases apoptosis. Furthermore, stable knockdown of ERbeta resulted in loss of growth contact inhibition. In this work, we report that loss of ERbeta is associated with a decrease of E-cadherin protein levels through different posttranscriptional regulatory mechanisms. Ligand activation of ERalpha induced E-cadherin extracellular shedding and internalization only in the absence of ERbeta, followed by lysosomal degradation. Loss of ERbeta also led to an increase of E-cadherin uptake in a ligand-independent manner through mechanisms that required caveolae formation. Proteasome activity was necessary for both mechanisms to operate. Increased E-cadherin internalization correlated with the up-regulation of beta-catenin transcriptional activity and impaired morphogenesis on Engelbreth-Holm-Swarm matrix. Taken together, these results emphasize the role of epithelial ERbeta in maintaining cell adhesion and a differentiated phenotype and highlight the potential importance of ERbeta for the design of specific agonists for use in breast cancer therapy.

Estrogen receptor beta inhibits angiogenesis and growth of T47D breast cancer xenografts.

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERalpha and ERbeta. ERalpha mediates the proliferative effect of estrogen in breast cancer cells, whereas ERbeta seems to be antiproliferative. We engineered ERalpha-positive T47D breast cancer cells to express ERbeta 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 ERbeta. The presence of ERbeta resulted in a reduction in tumor growth. Comparison of the ERbeta-expressing and non-ERbeta-expressing tumors revealed that the expression of ERbeta 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 beta (PDGFbeta). In cell culture, with the Tet-Off-regulated ERbeta-expressing cells, expression of ERbeta decreased expression of VEGF and PDGFbeta mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFbeta proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFbeta promoter constructs revealed a repressive effect of ERbeta at the promoter level of these genes. Taken together, these data show that introduction of ERbeta into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis.