Short carboxyl terminal parathyroid hormone peptides modulate human parathyroid hormone signaling in mouse osteoblasts.

BACKGROUND: Novel human parathyroid hormone (hPTH) peptides of unknown biological activity have recently been identified in the serum of subjects with normal renal function, chronic renal failure, and end-stage renal disease through the application of liquid chromatography-high resolution mass spectrometry. PURPOSE: of experiments: To determine the bioactivity of these peptides, we synthesized hPTH28-84, hPTH38-84, and hPTH45-84 peptides by solid phase peptide synthesis and tested their bioactivity in MC3T3-E1 mouse osteoblasts, either individually or together with the native hormone, hPTH1-84, by assessing the accumulation of 3´,5´-cyclic adenosine monophosphate (cAMP) and the induction of alkaline phosphatase activity. RESULTS: Increasing doses of hPTH1-84 (1-100 nM) increased the accumulation of cAMP and alkaline phosphatase activity in osteoblasts. hPTH28-84, hPTH38-84, and hPTH45-84 in concentrations of 1-100 nM were biologically inert. Surprisingly, 100 nM hPTH38-84 and hPTH45-84 increased the accumulation of cAMP in osteoblasts treated with increasing amounts of hPTH1-84. Human PTH28-84 had no effects on cAMP activity alone or in combination with hPTH1-84. Conversely, 100 nM hPTH38-84, hPTH45-84, and hPTH28-84 blocked the activation of alkaline phosphatase activity by hPTH1-84. CONCLUSIONS: The data show that the short carboxyl-terminal hPTH peptides, hPTH38-84 and hPTH45-84, increase the amount of cellular cAMP generated in cultured osteoblasts in response to treatment with full-length hPTH1-84 when compared to full-length hPTH1-84 alone. Human PTH28-84 had no effect on cAMP activity alone or in combination with hPTH1-84. Human PTH28-84, hPTH38-84 and hPTH45-84 reduced the effects of hPTH1-84 in osteoblasts with respect to the induction of alkaline phosphatase activity compared to hPTH1-84 alone. Short carboxyl peptides of human PTH are biologically inert but when administered together with full-length hPTH1-84 modulate the bioactivity of hPTH1-84 in osteoblasts.

Muscle-specific deletion of the vitamin D receptor in mice is associated with diaphragm muscle weakness.

Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma, and aging, are associated with an increased risk of pulmonary symptoms, longer duration of hospitalizations, and increasing requirements for mechanical ventilation. Vitamin D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knockout mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. Seven days following the final treatment, mice were euthanized, the DIAm was removed, and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx, and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.NEW & NOTEWORTHY Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

Impaired cardiac performance, protein synthesis, and mitochondrial function in tumor-bearing mice.

BACKGROUND: To understand the underlying mechanisms of cardiac dysfunction in cancer, we examined cardiac function, protein synthesis, mitochondrial function and gene expression in a model of heart failure in mice injected with Lewis lung carcinoma (LLC1) cells. EXPERIMENTAL DESIGN: Seven week-old C57BL/J6 male and female mice were injected with LLC1 cells or vehicle. Cardiac ejection fraction, ventricular wall and septal thickness were reduced in male, but not female, tumor-bearing mice compared to vehicle-injected control mice. Cardiac protein synthesis was reduced in tumor-bearing male mice compared to control mice (p = 0.025). Aspect ratio and form factor of cardiac mitochondria from the tumor-bearing mice were increased compared control mice (p = 0.042 and p = 0.0032, respectively) indicating a more fused mitochondrial network in the hearts of tumor-bearing mice. In cultured cardiomyocytes maximal oxygen consumption and mitochondrial reserve capacity were reduced in cells exposed to tumor cell-conditioned medium compared to non-conditioned medium (p = 0.0059, p = 0.0010). Whole transcriptome sequencing of cardiac ventricular muscle from tumor-bearing vs. control mice showed altered expression of 1648 RNA transcripts with a false discovery rate of less than 0.05. Of these, 54 RNA transcripts were reduced ≤ 0.5 fold, and 3 RNA transcripts were increased by ≥1.5-fold in tumor-bearing mouse heart compared to control. Notably, the expression of mRNAs for apelin (Apln), the apelin receptor (Aplnr), the N-myc proto-oncogene, early growth protein (Egr1), and the transcription factor Sox9 were reduced by >50%, whereas the mRNA for growth arrest and DNA-damage-inducible, beta (Gadd45b) is increased >2-fold, in ventricular tissue from tumor-bearing mice compared to control mice. CONCLUSIONS: Lung tumor cells induce heart failure in male mice in association with reduced protein synthesis, mitochondrial function, and the expression of the mRNAs for inotropic and growth factors. These data provide new mechanistic insights into cancer-associated heart failure that may help unlock treatment options for this condition.

Lung tumor cells inhibit bone mineralization and osteoblast activity.

Patients with non-small cell lung cancer (NSLC) often develop skeletal complications and fractures. To understand mechanisms of bone loss, we developed a murine model of non-metastatic NSLC. Decreased bone mineral density, trabecular thickness and mineralization, without an increase in bone resorption, were observed in vivo in mice injected with Lewis lung adenocarcinoma (LLC1) cells in the absence of tumor cell metastases. A decrease in trabecular bone mineral density was observed in mice injected with cell-free LLC1 CM. Plasma osteoblast biomarkers and PTH-related peptide (PTHrP) were reduced, and parathyroid hormone (PTH), 1,25-dihydroxyvitamin D, calcium and phosphate concentrations were normal in tumor-bearing mice. LLC1 cell conditioned medium (CM) inhibited alkaline phosphatase activity, osteoblast mineralization, and expression of Alpl and Ocn/Bglap mRNA in MC3T3 osteoblast cultures, whereas non-CM or CM from NIH/3T3 fibroblasts did not induce similar changes. LLC1 CM reduced Wnt3a-stimulated Tcf/Lef reporter plasmid activity and Wnt5A, Tcf1 and Lef1 mRNA expression in MC3T3 cells. Although concentrations of the Wnt inhibitor, DKK2, were increased in LLC1 CM compared to non-CM, depletion of DKK2 from LLC1 CM did not completely restore Wnt3a activity in MC3T3 cultures, and recombinant DKK2 failed to inhibit osteoblast mineralization. The data indicate that in a model of lung adenocarcinoma without bone metastases, tumor cells elaborate a secreted factor(s) that reduces bone mass, bone formation and osteoblast Wnt signaling without increases in bone resorption or calcium-regulating hormone concentrations. The factor(s) mediating this inhibition of osteoblast mineralization require further characterization.

1α,25-dihydroxyvitamin D3 mitigates cancer cell mediated mitochondrial dysfunction in human skeletal muscle cells.

Cancer cachexia is associated with muscle weakness and atrophy. We investigated whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), which has previously been shown to increase skeletal myoblast oxygen consumption rate, could reverse the deleterious effects of tumor cell conditioned medium on myoblast function. Conditioned medium from Lewis lung carcinoma (LLC1) cells inhibits oxygen consumption, increases mitochondrial fragmentation, inhibits pyruvate dehydrogenase activity, and enhances proteasomal activity in human skeletal muscle myoblasts. 1α,25(OH)2D3 reverses the tumor cell-mediated changes in mitochondrial oxygen consumption and proteasomal activity, without changing pyruvate dehydrogenase activity. 1α,25(OH)2D3 might be useful in treatment of weakness seen in association with CC.

Deletion of the intestinal plasma membrane calcium pump, isoform 1, Atp2b1, in mice is associated with decreased bone mineral density and impaired responsiveness to 1, 25-dihydroxyvitamin D3.

The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1(fl/fl)) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1(EKO) mice). Pmca1(EKO) mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) littermates. At two months of age, Pmca1(EKO) mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P < 0.037), and reduced femoral bone mineral density (P < 0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1(EKO) mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1(EKO) mice (P < 0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1(EKO) mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3.

The regulation of apoptosis by the downstream regulatory element antagonist modulator/potassium channel interacting protein 3 (DREAM/KChIP3) through interactions with hexokinase I.

The EF-hand protein, DREAM/KChIP3 (henceforth referred to as DREAM), regulates apoptosis by incompletely understood mechanisms. We demonstrate that in the presence of Ca2+, DREAM interacts with hexokinase I, a protein known to bind mitochondria and regulate apoptosis. A mutant DREAM protein construct incapable of binding Ca2+ does not associate with hexokinase I. The amino-terminal portion of DREAM is required for binding to hexokinase I, as a DREAM construct lacking the first 94 amino terminal residues fails to bind hexokinase I. Expression of DREAM in neuroblastoma cells enhances cisplatin mediated caspase-3 activity. Simultaneous expression of hexokinase I in such cells reduces DREAM-stimulated apoptosis. DREAM overexpression in neuroblastoma cells reduces hexokinase I localization on isolated mitochondria. The interaction of DREAM with hexokinase I may be important in the regulation of neuronal apoptosis.

Expression of sclerostin in the developing zebrafish (Danio rerio) brain and skeleton.

Sclerostin is a highly conserved, secreted, cystine-knot protein which regulates osteoblast function. Humans with mutations in the sclerostin gene (SOST), manifest increased axial and appendicular skeletal bone density with attendant complications. In adult bone, sclerostin is expressed in osteocytes and osteoblasts. Danio rerio sclerostin-like protein is closely related to sea bass sclerostin, and is related to chicken and mammalian sclerostins. Little is known about the expression of sclerostin in early developing skeletal or extra-skeletal tissues. We assessed sclerostin (sost) gene expression in developing zebrafish (D. rerio) embryos with whole mount is situ hybridization methods. The earliest expression of sost mRNA was noted during 12h post-fertilization (hpf). At 15 hpf, sost mRNA was detected in the developing nervous system and in Kupffer's vesicle. At 18, 20 and 22 hpf, expression in rhombic lip precursors was seen. By 24 hpf, expression in the upper and lower rhombic lip and developing spinal cord was noted. Expression in the rhombic lip and spinal cord persisted through 28 hpf and then diminished in intensity through 44 hpf. At 28 hpf, sost expression was noted in developing pharyngeal cartilage; expression in pharyngeal cartilage increased with time. By 48 hpf, sost mRNA was clearly detected in the developing pharyngeal arch cartilage. Sost mRNA was abundantly expressed in the pharyngeal arch cartilage, and in developing pectoral fins, 72, 96 and 120 hpf. Our study is the first detailed analysis of sost gene expression in early metazoan development.

The sclerostin-bone protein interactome.

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.

Sclerostin-erbB-3 interactions: modulation of erbB-3 activity by sclerostin.

To gain insights into the mechanism of action of sclerostin, a protein that regulates bone mass, we performed yeast two-hybrid analyses using human SOST (sclerostin) cDNA cloned into pGBKT7 DNA-binding domain vector as a bait, and a normalized, high-complexity, universal cDNA library in a GAL4 activating domain vector. We identified an interaction between sclerostin and the carboxyl-terminal portion of the receptor tyrosine-protein kinase erbB-3. To determine the biological relevance of this interaction, we treated MC3T3-E1 mouse osteoblast cells transfected with either a SOST expression plasmid or a control vector, with recombinant heregulin/neuregulin. Phospho-p44/42 (Thr202/Tyr204) MAPK was assessed in heregulin/neuregulin treated cells. We observed an increase in phospho-p44/42 (Thr202/Tyr204) MAPK concentrations in SOST transfected cells but not in cells transfected with a control vector, thus demonstrating a modulatory effect of sclerostin on heregulin/neuregulin signaling in osteoblasts. The data demonstrate that sclerostin functions in part, by modulating the activity of erbB-3.

Sclerostin binds and regulates the activity of cysteine-rich protein 61.

Sclerostin, a secreted glycoprotein, regulates osteoblast function. Using yeast two-hybrid and direct protein interaction analyses, we demonstrate that sclerostin binds the Wnt-modulating and Wnt-modulated, extracellular matrix protein, cysteine-rich protein 61 (Cyr61, CCN1), which regulates mesenchymal stem cell proliferation and differentiation, osteoblast and osteoclast function, and angiogenesis. Sclerostin was shown to inhibit Cyr61-mediated fibroblast attachment, and Cyr61 together with sclerostin increases vascular endothelial cell migration and increases osteoblast cell division. The data show that sclerostin binds to and influences the activity of Cyr61.

Secreted frizzled-related protein-4 reduces sodium-phosphate co-transporter abundance and activity in proximal tubule cells.

The phosphatonin, secreted frizzled-related protein-4 (sFRP-4), induces phosphaturia and inhibits 25-hydroxyvitamin D 1alpha-hydroxylase activity normally induced in response to hypophosphatemia. To determine the mechanism by which sFRP-4 alters renal phosphate (P(i)) transport, we examined the effect of sFRP-4 on renal brush border membrane (BBMV) Na(+)-dependent P(i) uptake, and the abundance and localization of the major Na(+)-P(i)-IIa co-transporter in proximal tubules and opossum kidney (OK) cells. Infusion of sFRP-4 increased renal fractional excretion of P(i) and decreased renal beta-catenin concentrations. The increase in renal P(i) excretion with sFRP-4 infusion was associated with a 21.9 +/- 3.4% decrease in BBMV Na(+)-dependent P(i) uptake (P < 0.001) compared with a 39.5 +/- 2.1% inhibition of Na(+)-dependent P(i) transport in renal BBMV induced by PTH (P < 0.001). sFRP-4 infusion was associated with a 30.7 +/- 4.8% decrease in Na(+)-P(i)-IIa co-transporter protein abundance (P < 0.01) assessed by immunoblotting methods compared to a 45.4 +/- 8.8% decrease induced by PTH (P < 0.001). In OK cells, sFRP-4 reduced surface expression of a heterologous Na(+)-P(i)-IIa co-transporter. We conclude that sFRP-4 increases renal P(i) excretion by reducing Na(+)-P(i)-IIa transporter abundance in the brush border of the proximal tubule through enhanced internalization of the protein.

Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent.

Tumors associated with osteomalacia elaborate the novel factor(s), phosphatonin(s), which causes phosphaturia and hypophosphatemia by cAMP-independent pathways. We show that secreted frizzled-related protein-4 (sFRP-4), a protein highly expressed in such tumors, is a circulating phosphaturic factor that antagonizes renal Wnt-signaling. In cultured opossum renal epithelial cells, sFRP-4 specifically inhibited sodium-dependent phosphate transport. Infusions of sFRP-4 in normal rats over 2 hours specifically increased renal fractional excretion of inorganic phosphate (FEPi) from 14% +/- 2% to 34% +/- 5% (mean +/- SEM, P < 0.01). Urinary cAMP and calcium excretion were unchanged. In thyro-parathyroidectomized rats, sFRP-4 increased FEPi from 0.7% +/- 0.2% to 3.8% +/- 1.2% (P < 0.05), demonstrating that sFRP-4 inhibits renal inorganic phosphate reabsorption by PTH-independent mechanisms. Administration of sFRP-4 to intact rats over 8 hours increased FEPi, decreased serum phosphate (1.95 +/- 0.1 to 1.53 +/- 0.09 mmol/l, P < 0.05) but did not alter serum 1alpha, 25-dihydroxyvitamin D, renal 25-hydroxyvitamin D 1alpha-hydroxylase cytochrome P450, and sodium-phosphate cotransporter mRNA concentrations. Infusion of sFRP-4 antagonizes Wnt action as demonstrated by reduced renal beta-catenin and increased phosphorylated beta-catenin concentrations. The sFRP-4 is detectable in normal human serum and in the serum of a patient with tumor-induced osteomalacia. Thus, sFRP-4 displays phosphatonin-like properties, because it is a circulating protein that promotes phosphaturia and hypophosphatemia and blunts compensatory increases in 1alpha, 25-dihydroxyvitamin D.

Characterization of a novel hexameric repeat DNA sequence in the promoter of the immediate early gene, IEX-1, that mediates 1alpha,25-dihydroxyvitamin D(3)-associated IEX-1 gene repression.

1alpha,25-Dihydroxyvitamin D(3)(1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D(3), mediates anti-proliferative effects in cells by regulating the expression of 1alpha,25(OH)(2)D(3)-responsive genes. The expression of the proliferation-promoting Immediate Early gene X-1 (IEX-1) is reduced by 1alpha,25(OH)(2)D(3) through unknown mechanisms. Here we report the presence of a novel inhibitory hexameric repeat DNA response element in the promoter of the human IEX-1 gene that mediates 1alpha,25(OH)(2)D(3)-associated IEX-1 gene repression. To localize a vitamin D sensitive DNA response element we transfected the keratinocyte-like cell line, HaCaT, (referred as HaCaT) with a series of plasmids containing full-length and truncated IEX-1 promoter elements fused to the luciferase reporter gene in the absence or presence of 1alpha,25(OH)(2)D(3), and we performed electrophoretic gel mobility assays in the presence of receptors for 1alpha,25(OH)(2)D(3) (vitamin D receptor, VDR) and 9-cis-retinoic acid (RXRalpha). We mapped a negative response element between nt -405 and -391(15 bp) of theIEX-1 promoter (5'-TGAACC AGG GAGTCA-3') that mediates transcriptional inhibition in response to 1alpha,25(OH)(2)D(3) and which requires expression of both nuclear receptors for 1alpha,25(OH)(2)D(3) and 9-cis-retinoic acid. Our data indicate that the physiological repression of IEX-1 gene expression by 1alpha,25(OH)(2)D(3) is directly mediated by nuclear VDR/RXRalpha heterodimers through a specific transcriptional element.