Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.     

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.     

Tumor-promoting phorbol esters and vanadate alter the sensitivity of HeLa S3 cells to poliovirus type 1

Treatment of HeLa S3 cells with tumor-promoting phorbol esters and vanadate increased their sensitivity to type 1 poliovirus. Since the sensitization could not be accounted for by increased virus binding or virus production, it appears that virus entry was facilitated by the treatments. When HeLa S3 cells were incubated with TPA for prolonged periods of time, they became resistant to poliovirus due to reduced ability to bind the virus.     

Selective inhibition of sodium-linked and sodium-independent bicarbonate/chloride antiport in Vero cells

The effects of compounds previously described to inhibit anion transport were tested for their ability to inhibit anion antiport in Vero cells as measured by uptake of 36Cl- by chloride self-exchange and as bicarbonate-linked uptake of 22Na+. While 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited both processes, ethacrynic acid and probenecid selectively inhibited the uptake of 36Cl-. Low concentrations of pyridoxal phosphate and picrylsulfonic acid selectively inhibited the bicarbonate linked uptake of 22Na+, while higher concentrations of these compounds also inhibited the uptake of 36Cl-. Measurements of the internal pH indicated that ethacrynic acid inhibits Na+-independent HCO-3/Cl- exchange, while it has no measurable effect on Na+-linked bicarbonate-dependent regulation of the internal pH. Conversely, picrylsulfonic acid selectively inhibits the latter process. The results indicate that anion antiport in Vero cells occurs by two independent processes.     

Effect of intracellular pH on the rate of chloride uptake and efflux in different mammalian cell lines

The effect of the internal pH on the rate of 36Cl- uptake and efflux was measured. While the rate of 36Cl- uptake by the antiport increased almost 10-fold over a narrow pH range in a number of cell lines, in other cells the pH-induced increase was considerably less. With increasing pH, the rate of 36Cl- efflux into chloride-free buffers increased strongly over a narrow pH range in all cell lines studied. Two groups of cell lines appeared, one group where the half-maximal increase in uptake and efflux rate was at pH 7.0-7.1 and another group where the corresponding values were 0.2-0.4 pH unit higher. The stoichiometry between chloride influx and chloride-stimulated efflux was close to 1:1 under various conditions.     

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragment. Effect on membrane translocation and on formation of cation channels.

Diphtheria toxin B fragment is capable of forming cation-selective channels in the plasma membrane. Such channels may be involved in the translocation of the toxin A fragment to the cytosol. Seven negatively charged amino acids in the B fragment were replaced one by one by lysines, followed by studies of cytotoxicity and channel-forming ability of the different mutants. The mutant D392K showed a strong reduction in binding to cell surface receptors. Of the six mutants that showed wild-type binding affinity, the two mutants D295K and D318K were very inefficient in forming channels. These two mutants had the lowest ability to mediate A fragment translocation. The mutant E362K was able both to induce cation channel formation and to mediate A fragment translocation at a higher pH value than the wild-type B fragment. The results support the notion that formation of cation channels is of importance for the translocation of the A fragment across the plasma membrane, and they indicate that the pH requirement for translocation of the A fragment to the cytosol is partly determined by the B fragment.