Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH) vitamin D metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH) vitamin D, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH) vitamin D are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24 h) C-terminal FGF23 but iFGF23 only after 4 days without effects on PTH and plasma phosphate. 1,25 (OH) D levels and αklotho expression in the kidney decrease after 4 days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71 erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH) vitamin D levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism.

The gene coding for the human homologue of the Drosophila segment polarity gene patched (PTCH1) is mutated in several common human tumors. In mice, haplodeficiency at the Ptch1 locus results in severe histologic defects in mammary ductal structure. We found no mutations within the coding region of PTCH1 in 17 human primary breast carcinomas. However, the biallelic Pro1315Leu (C3944T ) polymorphism of PTCH1 was significantly associated with breast cancer in 41 Bavarian patients compared to 85 healthy controls. We investigated whether this variant influences susceptibility for breast cancer in 611 breast cancer patients diagnosed by age 50 years and 1,057 controls matched by age and study region in Germany and in 1,093 breast cancer patients from the United Kingdom. Allele and genotype frequencies were not different between cases and controls. However, multivariate logistic regression analysis revealed an effect modification of oral contraceptive use (OC) on breast cancer risk by Leu-carrier status. Compared to women who have Pro/Pro and never used OC, Pro/Pro OC users had an increased odds ratio for breast cancer of 1.7. The odds ratio was also 1.7 for Leu-carriers who never used OC, but this was attenuated among Leu-carriers who ever used OC by 20%. Key words: genetic polymorphism; breast cancer; gene-environment interaction; oral contraceptivesThe tumor suppressor gene PTCH1 is a downstream receptor in the hedgehog family of cell signaling proteins and plays an essential role in many aspects of cell growth and cell differentiation. Germline mutations in PTCH1 have been detected in patients with nevoid basal cell carcinoma syndrome (NBCCS), in which patients are predisposed to developmental abnormalities and a variety of neoplasms including basal cell carcinoma and medulloblastoma. 1,2 In addition, somatic mutations in PTCH1 have been identified in sporadic cases of basal cell carcinomas and of medulloblastoma and in a variety of other tumors (see review). 3,4 Interestingly, nonsense mutations in PTCH1 have been reported in 2 of 7 breast carcinomas, 5 suggesting an involvement of the SHH/PTCH1 signaling pathway in development of this kind of tumor. Although a similar study in a larger sample of 45 breast carcinomas did not reveal any inactivating PTCH1 mutations, 6 the possible involvement of this gene in the formation of breast carcinoma has also been demonstrated in an animal model for Ptch1 haplodeficiency. 7 All postpubescent virgin mice heterozygous for Ptch1 developed ductal hyperplasia and dysplasia of the mammary glands, which reverted during late pregnancy and lactation but returned upon involution and gland remodeling. Treatment with estradiol and progesterone enhanced the mutant histologic phenotype. These data suggest a role for the Ptch1 signaling network in mammary growth and differentiation.To obtain further insights into the involvement of PTCH1 in breast carcinoma formation, we analyzed a series of breast carcinomas for PTCH1 mutations. In the process we observed that the biallelic Pro1315Leu (...

Inherited mutations of Patched (PTCH) in the nevoid basal cell carcinoma syndrome (NBCCS) lead to several developmental defects and contribute to tumor formation in a variety of tissues. PTCH mutations have been also identified in sporadic tumors associated with NBCCS including basal cell carcinoma (BCC) and medulloblastoma. Mice heterozygous for Ptch recapitulate the typical developmental symptoms of NBCCS and develop rhabdomyosarcoma (RMS) and medulloblastoma. PTCH is assumed to act as a tumor suppressor gene although inactivation of both alleles has been demonstrated only in a fraction of tumors. We have investigated the status of Ptch in RMS of heterozygous Ptch neo67/+ mice. Although the wild-type Ptch allele was retained in tumor tissue, the high levels of Ptch mRNA in these tumors result from overexpression of the mutant Ptch transcript. Our results suggest that the wild-type Ptch allele might be selectively silenced in RMS tissue or, alternatively, that haploinsufficiency of Ptch is sufficient to promote RMS formation in mice.

Mutations in the human homologue of Drosophila Patched1 (PTCH1) have been found in several common tumours including basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (RMS). Medulloblastoma and RMS are also present in the murine model for Ptch1 deficiency. Tumours in heterozygous Ptch1(neo67/+) mice consistently exhibit elevated transcript levels of the proto-oncogene Gli1, of Ptch1 itself, and of the insulin-like growth factor 2 (Igf2). The present study has investigated additional molecular changes in RMSs of Ptch1 mutant mice by means of microarray analysis and protein expression analysis. The data show activation of the cell survival-promoting Akt/protein kinase B (Pkb). Furthermore, RMSs express increased levels of the anti-apoptotic protein Bcl-2 and of genes and proteins known to inhibit cell proliferation, including Gadd45a and p27kip1. Taken together, the data suggest that the formation of RMSs in Ptch1 mutants is associated with the ability of tumour cells to resist apoptosis.

BackgroundThe 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) together with parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) regulates calcium (Ca2+) and phosphate (Pi) homeostasis, 1,25(OH)2D3 synthesis is mediated by hydroxylases of the cytochrome P450 (Cyp) family. Vitamin D is first modified in the liver by the 25-hydroxylases CYP2R1 and CYP27A1 and further activated in the kidney by the 1α-hydroxylase CYP27B1, while the renal 24-hydroxylase CYP24A1 catalyzes the first step of its inactivation. While the kidney is the main organ responsible for circulating levels of active 1,25(OH)2D3, other organs also express some of these enzymes. Their regulation, however, has been studied less.Methods and resultsHere we investigated the effect of several Pi-regulating factors including dietary Pi, PTH and FGF23 on the expression of the vitamin D hydroxylases and the vitamin D receptor VDR in renal and extrarenal tissues of mice. We found that with the exception of Cyp24a1, all the other analyzed mRNAs show a wide tissue distribution. High dietary Pi mainly upregulated the hepatic expression of Cyp27a1 and Cyp2r1 without changing plasma 1,25(OH)2D3. FGF23 failed to regulate the expression of any of the studied hydroxylases at the used dosage and treatment length. As expected, renal mRNA expression of Cyp27b1 was reduced and Cyp24a1 was increased in response to 1,25(OH)2D3 treatment. However, the 25-hydroxylases were rather unaffected by 1,25(OH)2D3 treatment.ConclusionsThe analyzed vitamin D hydroxylases are regulated in a tissue and treatment-specific manner.

NaPi-IIb/Slc34a2 is a Na+-dependent phosphate transporter that accounts for the majority of active phosphate transport into intestinal epithelial cells. Its abundance is regulated by dietary phosphate, being high during dietary phosphate restriction. Intestinal ablation of NaPi-IIb in mice leads to increased fecal excretion of phosphate, which is compensated by enhanced renal reabsorption. Here we compared the adaptation to dietary phosphate of wild type (WT) and NaPi-IIb−/− mice. High phosphate diet (HPD) increased fecal and urinary excretion of phosphate in both groups, though NaPi-IIb−/− mice still showed lower urinary excretion than WT. In both genotypes low dietary phosphate (LDP) resulted in reduced fecal excretion and almost undetectable urinary excretion of phosphate. Consistently, the expression of renal cotransporters after prolonged LDP was similar in both groups. Plasma phosphate declined more rapidly in NaPi-IIb−/− mice upon LDP, though both genotypes had comparable levels of 1,25(OH)2vitamin D3, parathyroid hormone and fibroblast growth factor 23. Instead, NaPi-IIb−/− mice fed LDP had exacerbated hypercalciuria, higher urinary excretion of corticosterone and deoxypyridinoline, lower bone mineral density and higher number of osteoclasts. These data suggest that during dietary phosphate restriction NaPi-IIb-mediated intestinal absorption prevents excessive demineralization of bone as an alternative source of phosphate.

Key points Intestinal absorption of phosphate proceeds via an active/transcellular route mostly mediated by NaPi‐IIb/Slc34a2 and a poorly characterized passive/paracellular pathway. Intestinal phosphate absorption and expression of NaPi‐IIb are stimulated by 1,25(OH)2 vitamin D3 but whether NaPi‐IIb is the only target under hormonal control remains unknown. We report that administration of 1,25(OH)2 vitamin D3 to wild‐type mice resulted in the expected increase in active transport of phosphate in jejunum, without changing paracellular fluxes. Instead, the same treatment failed to alter phosphate transport in intestinal‐depleted Slc34a2‐deficient mice. In both genotypes, 1,25(OH)2 vitamin D3 induced similar hyperphosphaturic responses and changes in the plasma levels of FGF23 and PTH. While urinary phosphate loss induced by administration of 1,25(OH)2 vitamin D3 did not alter plasma phosphate, further studies should investigate whether chronic administration would lead to phosphate imbalance in mice with reduced active intestinal absorption. Abstract Intestinal absorption of phosphate is stimulated by 1,25(OH)2 vitamin D3. At least two distinct mechanisms underlie phosphate absorption in the gut, an active transcellular transport requiring the Na+/phosphate cotransporter NaPi‐IIb/Slc34a2, and a poorly characterized paracellular passive pathway. 1,25(OH)2 vitamin D3 stimulates NaPi‐IIb expression and function, and loss of NaPi‐IIb reduces intestinal phosphate absorption. However, it is remains unknown whether NaPi‐IIb is the only target for hormonal regulation by 1,25(OH)2 vitamin D3. Here we compared the effects of intraperitoneal administration of 1,25(OH)2 vitamin D3 (2 days, once per day) in wild‐type and intestinal‐specific Slc34a2‐deficient mice, and analysed trans‐ vs. paracellular routes of phosphate absorption. We found that treatment stimulated active transport of phosphate only in jejunum of wild‐type mice, though NaPi‐IIb protein expression was upregulated in jejunum and ileum. In contrast, 1,25(OH)2 vitamin D3 administration had no effect in Slc34a2‐deficient mice, suggesting that the hormone specifically regulates NaPi‐IIb expression. In both groups, 1,25(OH)2 vitamin D3 elicited the expected increase of plasma fibroblast growth factor 23 (FGF23) and reduction of parathyroid hormone (PTH). Treatment resulted in hyperphosphaturia (and hypercalciuria) in both genotypes, though mice remained normophosphataemic. While increased intestinal absorption and higher FGF23 can trigger the hyperphosphaturic response in wild types, only higher FGF23 can explain the renal response in Slc34a2‐deficient mice. Thus, 1,25(OH)2 vitamin D3 stimulates intestinal phosphate absorption by acting on the active transcellular pathway mostly mediated by NaPi‐IIb while the paracellular pathway appears not to be affected.

Aims Dietary inorganic phosphate (Pi) modulates renal Pi reabsorption by regulating the expression of the NaPi‐IIa and NaPi‐IIc Pi transporters. Here, we aimed to clarify the role of several Pi‐regulatory mechanisms including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and inositol hexakisphosphate kinases (IP6‐kinases) in the acute regulation of NaPi‐IIa and NaPi‐IIc. Methods Wildtype (WT) and PTH‐deficient mice (PTH‐KO) with/without inhibition of FGF23 signalling were gavaged with Pi/saline and examined at 1, 4 and 12 h. Results Pi‐gavage elevated plasma Pi and decreased plasma Ca2+ in both genotypes after 1 h Within 1 h, Pi‐gavage decreased NaPi‐IIa abundance in WT and PTH‐KO mice. NaPi‐IIc was downregulated 1 h post‐administration in WT and after 4 h in PTH‐KO. PTH increased after 1 h in WT animals. After 4 h Pi‐gavage, FGF23 increased in both genotypes being higher in the KO group. PTHrp and dopamine were not altered by Pi‐gavage. Blocking FGF23 signalling blunted PTH upregulation in WT mice and reduced NaPi‐IIa downregulation in PTH‐KO mice 4 h after Pi‐gavage. Inhibition of IP6‐kinases had no effect. Conclusions (1) Acute downregulation of renal Pi transporters in response to Pi intake occurs also in the absence of PTH and FGF23 signalling, (2) when FGF23 signalling is blocked, a partial contribution of PTH is revealed, (3) IP6 kinases, intracellular Pi‐sensors in yeast and bacteria, are not involved, and (4) Acute Pi does not alter PTHrp and dopamine. Thus, signals other than PTH, PTHrp, FGF23 and dopamine contribute to renal adaption.