Fibroblast growth factor 23: the making of a hormone.

Fibroblast growth factor 23 (FGF23) modulates serum phosphate and 1alpha,25-dihydroxyvitamin D3 levels. FGF23 expression is in turn regulated by 1alpha,25-dihydroxyvitamin D3 and dietary phosphate load, and is strikingly elevated during renal progression. In this issue, Nagano and colleagues report that FGF23 can be modulated by intermittently high dietary phosphate in the presence of compromised renal function.

FGF-23 inhibits renal tubular phosphate transport and is a PHEX substrate.

Oncogenic osteomalacia (OOM), X-linked hypophosphatemia (XLH), and autosomal dominant hypophosphatemic rickets (ADHR) are phenotypically similar disorders characterized by hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum calcitriol concentrations, normal serum concentrations of calcium and parathyroid hormone, and defective skeletal mineralization. XLH results from mutations in the PHEX gene, encoding a membrane-bound endopeptidase, whereas ADHR is associated with mutations of the gene encoding FGF-23. Recent evidence that FGF-23 is expressed in mesenchymal tumors associated with OOM suggests that FGF-23 is responsible for the phosphaturic activity previously termed "phosphatonin." Here we show that both wild-type FGF-23 and the ADHR mutant, FGF-23(R179Q), inhibit phosphate uptake in renal epithelial cells. We further show that the endopeptidase, PHEX, degrades native FGF-23 but not the mutant form. Our results suggest that FGF-23 is involved in the pathogenesis of these three hypophosphatemic disorders and directly link PHEX and FGF-23 within the same biochemical pathway.

Ability of adenovirus vectors containing different CFTR transcriptional cassettes to correct ion transport defects in CF cells.

Cystic fibrosis (CF) airway epithelial cells exhibit defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated chloride (Cl) secretion, abnormal hyperabsorption of sodium (Na+), and aberrant fluid transport. Adenovirus-mediated transduction of cystic fibrosis transmembrane conductance regulator (CFTR) corrects these ion and fluid transport abnormalities in CF cells. However, several challenges remain pertaining to the use of adenovirus vectors for gene delivery, including the efficiency of gene transfer and the host response to the vector. To improve the efficacy of adenovirus-mediated gene transfer, we have constructed a series of recombinant adenoviruses containing different CFTR transcriptional units, and we have evaluated their relative ability to correct electrolyte and fluid transport in polarized CF airway epithelial cells. The ability of the vectors to correct the CF Cl- transport defects was greatest when the human cytomegalovirus promoter was used. The E1a and phosphoglycerate kinase promoters resulted in the greatest persistence of functional CFTR expression. Efficacy of gene expression by recombinant adenoviruses improved as the cells were treated with increasing multiplicities of infection, as the duration of viral contact with the target cells was lengthened, and when the virus concentration was increased. Transduction of functional CFTR Cl- channel activity reversed the abnormal Na+ hyperabsorption observed in CF cells in a dose-dependent manner, suggesting that Na+ channel activity is downregulated by CFTR. Although efficient correction of both cAMP-mediated Cl- transport and fluid secretion could be achieved readily with these vectors, normalization of the Na+ absorption required vector administration at high multiplicities of infection.

Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells.

An inducible gene amplification system was utilized to study the effects of overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) in vitro. BTS, a monkey kidney cell line expressing a temperature-sensitive simian virus 40 (SV-40) large T antigen was stably transfected at the nonpermissive temperature with a plasmid containing an SV-40 origin of replication and the cDNA for either the wild-type CFTR or the mutant G551D-CFTR. Shift of the isolated cell lines to the permissive temperature resulted in induction and accumulation to high levels of the CFTR plasmid, mRNA, and protein. However, high-level expression of CFTR was transient in both BTS-CFTR and BTS-G551D cells due to a decrease in their respective levels of CFTR mRNA. Because G551D-CFTR only exhibits residual Cl channel activity, this suggests that the observed downregulation with BTS-G551D cells may have been induced by either the physical presence of high amounts of CFTR or some low threshold level of Cl- channel activity. Examination of cell growth properties revealed a correlation between high-level expression of wild-type CFTR and growth arrest of the cells at the G2/M phase. However, similar induction of the G551D-CFTR mutant showed only a slight growth inhibition and little enrichment of cells at the G2/M phase. Cytofluorographic analysis further revealed that BTS-CFTR cells were significantly larger than parental BTS or BTS-G551D cells at all stages of the cell cycle. These results indicate that CFTR overexpression is capable of inducing its own downregulation and that high levels of Cl- channel activity can result in increased cell volume and subsequent cell growth abnormalities.

Functional activation of the cystic fibrosis trafficking mutant delta F508-CFTR by overexpression.

The most common mutation in the gene associated with cystic fibrosis (CF) causes deletion of phenylalanine at residue 508 (delta F508) of the gene product called CFTR. This mutation results in the synthesis of a variant CFTR protein that is defective in its ability to traffic to the plasma membrane. Because earlier studies showed delta F508-CFTR retains significant phosphorylation-regulated chloride (Cl-) channel activity, processes capable of restoring the mislocalized delta F508-CFTR to the correct cellular destination may have therapeutic benefit. Here we report one such process that involves overexpression of the mutant protein and appears to result in the escape of a small amount of delta F508-CFTR to the plasma membrane. In recombinant cells where expression of delta F508-CFTR is controlled by the metallothionein promoter, this effect can be brought about by treatment with sodium butyrate. Although cAMP-activated Cl- channel activity could also be detected in immortalized human airway epithelial cells homozygous for the delta F508 mutation at the single cell level, treatment with butyrate did not generate a measurable cAMP-stimulated Cl- current in polarized monolayers of primary CF airway epithelia. However, the observation that overexpression can effect the presence of recombinant delta F508-CFTR at the plasma membrane suggests that perhaps other butyrate-like compounds that are more potent and more specific for the promoter of the CF gene may be efficacious in alleviating the Cl- channel defect associated with CF.

Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation.

The c-fos proto-oncogene transcript is one of the most labile mammalian mRNAs known. Rapid degradation of c-fos mRNA is mediated by both the c-fos protein-coding region and an AU-rich element in the 3'-untranslated region. Here we present evidence that the c-fos coding region contains multiple destabilizing elements that can function independently to facilitate both deadenylation and decay of mRNA. The ability of these coding region destabilizing elements to direct deadenylation and decay requires the assembly of ribosomes at the 5' end of this domain and, most likely, translation of the message.

Hormone regulated enzyme activities of plasma membrane of decidual endometrium of the rat.

Plasma membranes were purified from deciduoma of pseudopregnant rats and rat liver. Preparations contained 80% plasma membrane-derived material as based on electron microscope morphometry and analysis of enzyme markers. Several plasma membrane enzymes were tested for direct response to hormones. NADH-ferricyanide reductase of plasma membranes from both tissues was stimulated by glucagon and inhibited by insulin but was unresponsive to steroids. For steroids, responsiveness was limited to a reduction in NaF-stimulated adenylate cyclase activity by the steroid R5020. Thus, interaction of steroid hormones with plasma membranes, unlike that of glucagon and insulin, is not reflected in an altered activity of plasma membrane-bound dehydrogenases but may be exerted directly on adenylate cyclase.

Steroid interactions with plasma membrane of decidual endometrium of the rat.

Plasma membranes were purified from deciduoma of pseudopregnant rats, rat liver and intestine, and calf uterus. Steroid binding evaluated with deciduoma plasma membranes showed competitive progestin binding, in contrast with estradiol binding which was nondisplaceable as measured by competition binding assay. When the photosensitive steroid [3H]-R5020 was photocrosslinked to plasma membrane, binding was reduced competitively by either progesterone or R5020. These results indicate that the decidual cell plasma membrane contains specific sites for interactions with progestins.

myc and E1A oncogenes alter the responses of PC12 cells to nerve growth factor and block differentiation.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc or E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors and ornithine decarboxylase induction were similar in myc- and E1A-expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors and hormones.