Publications by authors named "Theodore A Craig"

34 Publications

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

Biochem Biophys Res Commun 2021 Oct 28;572:15-19. Epub 2021 Jul 28.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic Rochester, 200 First Street Southwest, Rochester, MN, 55905, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, 200 First Street Southwest, Rochester, MN, 55905, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2021.07.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380684PMC
October 2021

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ.

J Biol Chem 2021 Aug 24;297(2):100912. Epub 2021 Jun 24.

Nephrology and Hypertension Research, Division of Hypertension and Nephrology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA. Electronic address:

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.
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http://dx.doi.org/10.1016/j.jbc.2021.100912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319531PMC
August 2021

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

J Appl Physiol (1985) 2021 07 20;131(1):95-106. Epub 2021 May 20.

Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.

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 D. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D 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 mice (+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. + mice treated with vehicle and 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. 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.
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http://dx.doi.org/10.1152/japplphysiol.00194.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325609PMC
July 2021

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

PLoS One 2019 18;14(12):e0226440. Epub 2019 Dec 18.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226440PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919625PMC
March 2020

Lung tumor cells inhibit bone mineralization and osteoblast activity.

Biochem Biophys Res Commun 2019 11 16;519(3):566-571. Epub 2019 Sep 16.

Department of Medicine, Divisions of Nephrology and Hypertension and Endocrinology, Diabetes, Metabolism and Nutrition, Department of Biochemistry and Molecular Biology, Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA; Nephrology and Hypertension and Endocrinology, Diabetes, Metabolism and Nutrition, Department of Biochemistry and Molecular Biology, Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA; Endocrinology, Diabetes, Metabolism and Nutrition, Department of Biochemistry and Molecular Biology, Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA; Department of Biochemistry and Molecular Biology, Department of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879054PMC
November 2019

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

Biochem Biophys Res Commun 2018 02;496(2):746-752

Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Division of Nephrology and Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Division of Endocrinology/Metabolism, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Electronic address:

Cancer cachexia is associated with muscle weakness and atrophy. We investigated whether 1α,25-dihydroxyvitamin D (1α,25(OH)D), 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)D reverses the tumor cell-mediated changes in mitochondrial oxygen consumption and proteasomal activity, without changing pyruvate dehydrogenase activity. 1α,25(OH)D might be useful in treatment of weakness seen in association with CC.
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http://dx.doi.org/10.1016/j.bbrc.2018.01.092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812288PMC
February 2018

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

Biochemistry 2017 07 3;56(28):3523-3530. Epub 2017 Jul 3.

Department of Chemistry, Washington University in St. Louis , St. Louis, Missouri 63130, United States.

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca-binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca but detaches from DRE under Ca stimulation, allowing gene expression. The Ca binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca. Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca-binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca signal to CREB-mediated gene transcription.
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http://dx.doi.org/10.1021/acs.biochem.7b00100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551683PMC
July 2017

1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells.

J Biol Chem 2016 Jan 24;291(3):1514-28. Epub 2015 Nov 24.

From the Departments of Medicine, Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength.
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http://dx.doi.org/10.1074/jbc.M115.684399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714233PMC
January 2016

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.

Biochem Biophys Res Commun 2015 Nov 21;467(1):152-6. Epub 2015 Sep 21.

Nephrology Research, Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2015.09.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772868PMC
November 2015

Alterations in vitamin D metabolite, parathyroid hormone and fibroblast growth factor-23 concentrations in sclerostin-deficient mice permit the maintenance of a high bone mass.

J Steroid Biochem Mol Biol 2015 Apr 22;148:225-31. Epub 2014 Nov 22.

Division of Nephrology and Hypertension, Mayo Clinic, 200 1st St., SW, Rochester, MN 55905, USA; Department of Internal Medicine, Mayo Clinic, 200 1st St., SW, Rochester, MN 55905 USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st St., SW, Rochester, MN 55905, USA. Electronic address:

Humans with mutations of the sclerostin (SOST) gene, and knockout animals in which the Sost gene has been experimentally deleted, exhibit an increase in bone mass. We review the mechanisms by which Sost knockout mice are able to accrete increased amounts of calcium and phosphorus required for the maintenance of a high bone mass. Recently published information from our laboratory, shows that bone mass is increased in Sost-deficient mice through an increase in osteoblast and a decrease in osteoclast activity, which is mediated by activation of β-catenin and an increase in prostacyclin synthesis in osteocytes and osteoblasts. The increases in calcium and phosphorus retention required for enhanced bone mineral accretion are brought about by changes in the vitamin D endocrine system, parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23). Thus, in Sost knockout mice, concentrations of serum 1,25-dihydroxyvitamin D (1,25(OH)2D) are increased and concentrations of FGF-23 are decreased thereby allowing a positive calcium and phosphorus balance. Additionally, in the absence of Sost expression, urinary calcium is decreased, either through a direct effect of sclerostin on renal calcium handling, or through its effect on the synthesis of 1,25(OH)2D. Adaptations in vitamin D, PTH and FGF-23 physiology occur in the absence of sclerostin expression and mediate increased calcium and phosphorus retention required for the increase in bone mineralization. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.
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http://dx.doi.org/10.1016/j.jsbmb.2014.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361263PMC
April 2015

Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone.

Biochem Biophys Res Commun 2014 May 26;448(1):83-8. Epub 2014 Apr 26.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Electronic address:

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.
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http://dx.doi.org/10.1016/j.bbrc.2014.04.092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052706PMC
May 2014

Detection of 1α,25-dihydroxyvitamin D-regulated miRNAs in zebrafish by whole transcriptome sequencing.

Zebrafish 2014 Jun 20;11(3):207-18. Epub 2014 Mar 20.

1 Nephrology and Hypertension Research, Department of Internal Medicine, Mayo Clinic , Rochester, Minnesota.

The sterol hormone, 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃), regulates gene expression and messenger RNA (mRNA) concentrations in zebrafish in vivo. Since mRNA concentrations and translation are influenced by micro-RNAs (miRNAs), we examined the influence of 1α,25(OH)₂D₃ on miRNA expression in zebrafish in vivo with whole transcriptome RNA sequencing, searched for miRNA binding sites in 1α,25(OH)₂D₃-sensitive genes, and performed correlation analyses between 1α,25(OH)₂D₃-sensitive miRNAs and mRNAs. In vehicle- and 1α,25(OH)₂D₃-treated, 7-day postfertilization larvae, between 282 and 295 known precursor miRNAs were expressed, and in vehicle- and 1α,25(OH)₂D₃-treated fish, between 83 and 122 novel miRNAs were detected. Following 1α,25(OH)₂D₃ treatment, 31 precursor miRNAs were differentially expressed (p<0.05). The differentially expressed miRNAs are predicted to potentially alter mRNAs for metabolic enzymes, transcription factors, growth factors, and Jak-STAT signaling. We verified the role of a 1α,25(OH)₂D₃-sensitive miRNA, miR125b, by demonstrating alterations in the concentrations of the mRNA of a 1α,25(OH)₂D₃-regulated gene, Cyp24a1, following transfection of renal cells with a miR125b miRNA mimic. Changes in the Cyp24a1 mRNA concentration by the miR125b miRNA mimic were associated with changes in the protein for Cyp24a1. Our data show that 1α,25(OH)₂D₃ regulates miRNA in zebrafish larvae in vivo and could thereby influence vitamin D-sensitive mRNA concentrations.
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http://dx.doi.org/10.1089/zeb.2013.0899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050706PMC
June 2014

Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium.

Proc Natl Acad Sci U S A 2013 Apr 25;110(15):6199-204. Epub 2013 Mar 25.

Nephrology Research, Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost(-/-)) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion.
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http://dx.doi.org/10.1073/pnas.1221255110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625358PMC
April 2013

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

Biochem Biophys Res Commun 2013 Apr 21;433(4):508-12. Epub 2013 Mar 21.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

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.
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http://dx.doi.org/10.1016/j.bbrc.2013.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3640266PMC
April 2013

The potassium channel interacting protein 3 (DREAM/KChIP3) heterodimerizes with and regulates calmodulin function.

J Biol Chem 2012 Nov 27;287(47):39439-48. Epub 2012 Sep 27.

Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota 55905, USA.

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.
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http://dx.doi.org/10.1074/jbc.M112.398495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501030PMC
November 2012

Research resource: whole transcriptome RNA sequencing detects multiple 1α,25-dihydroxyvitamin D(3)-sensitive metabolic pathways in developing zebrafish.

Mol Endocrinol 2012 Sep 25;26(9):1630-42. Epub 2012 Jun 25.

Nephrology and Hypertension Research, Medical Sciences, Mayo Clinic, Rochester, Minnesota 55905, USA.

The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3))].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)(2)D(3)-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)(2)D(3). Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)(2)D(3) vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)(2)D(3)-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)(2)D(3) functions in vivo in developing eukaryotes.
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http://dx.doi.org/10.1210/me.2012-1113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434529PMC
September 2012

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

Gene Expr Patterns 2012 Aug-Sep;12(7-8):228-35. Epub 2012 May 7.

Division of Nephrology and Hypertension, Mayo Clinic, 200 1st St., Southwest, Rochester, MN 55905, USA.

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.
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http://dx.doi.org/10.1016/j.gep.2012.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435489PMC
January 2013

The sclerostin-bone protein interactome.

Biochem Biophys Res Commun 2012 Jan 22;417(2):830-5. Epub 2011 Dec 22.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

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.
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http://dx.doi.org/10.1016/j.bbrc.2011.12.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259242PMC
January 2012

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

Biochem Biophys Res Commun 2010 Nov 14;402(2):421-4. Epub 2010 Oct 14.

Nephrology and Hypertension Research, Department of Internal Medicine, MS 1-120, Mayo Clinic, 200 1st St., Southwest, Rochester, MN 55905, USA.

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.
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http://dx.doi.org/10.1016/j.bbrc.2010.10.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2992958PMC
November 2010

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

Biochem Biophys Res Commun 2010 Jan 31;392(1):36-40. Epub 2009 Dec 31.

Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

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.
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http://dx.doi.org/10.1016/j.bbrc.2009.12.143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2821021PMC
January 2010

Production and characterization of monoclonal antibodies to human sclerostin.

Hybridoma (Larchmt) 2009 Oct;28(5):377-81

Nephrology Research, Department of Medicine, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.

We developed and characterized monoclonal antibodies directed against the amino-terminal and carboxy-terminal regions of human and mouse sclerostin (scl). Amino-terminal and carboxy-terminal scl peptides with limited homology to scl domain-containing protein-1 were synthesized using f-moc chemistry. The peptides were conjugated to keyhole limpet hemocyanin and the conjugates were used for immunization of mice. Monoclonal antibodies were obtained and characterized using bacterially expressed and insect cell-expressed recombinant scl. The amino-terminal (IgG 2aK) and carboxy-terminal (IgG 2bK) antibodies bound bioactive sclerostin that was expressed in an insect-cell expression system with dissociation constants in the nanomolar range. The antibodies are potentially useful agents that can be used for modulating sclerostin bioactivity.
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http://dx.doi.org/10.1089/hyb.2009.0036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145175PMC
October 2009

Expression and regulation of the vitamin D receptor in the zebrafish, Danio rerio.

J Bone Miner Res 2008 Sep;23(9):1486-96

Nephrology and Hypertension Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.

Vitamin D and vitamin D metabolites such as 25-hydroxyvitamin D and 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)(2)D(3)] circulate in the serum of fish. The receptor for 1alpha,25(OH)(2)D(3) (VDR) has previously been cloned from fish intestine, and ligand binding assays have shown the presence of the VDR in the gills, intestine, and liver of fish. Using immunohistochemical methods with specific antibodies against the VDR, we now report that the VDR is widely expressed in tissues of the adult male and female zebrafish, Danio rerio, specifically in epithelial cells of gills, tubular cells of the kidney, and absorptive cells in the intestine. Additionally, the VDR is expressed in the skin, the olfactory organ, the retina, brain, and spinal cord. Sertoli cells of the testis, oocytes, acinar cells of the pancreas, hepatocytes, and bile duct epithelial cells express substantial amounts of the receptor. Osteoblast-like cells and chondrocytes also express VDR. Preimmune serum and antiserum preadsorbed with Danio VDR protein fails to detect VDR in the same tissues. The VDR is also present in the developing eye, brain, and otic vesicle of 48- and 96-h postfertilization zebrafish embryos. Parenteral administration of 1alpha,25(OH)(2)D(3) increases concentrations of VDR in intestinal epithelial cells but not in epithelial cells of the gills. Lithocholic acid, however, does not alter concentrations of VDR after parenteral administration. The data suggest that VDR is widely distributed in tissues of the zebrafish, D. rerio, and is likely to play important roles in epithelial transport, bone, and endocrine function. Furthermore, concentrations of the receptor seem to be regulated by its ligand, 1alpha,25-dihydroxyvitamin D but not by lithocholic acid. Zebrafish may serve as a useful model in which to assess the function of the VDR in diverse tissues.
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http://dx.doi.org/10.1359/jbmr.080403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2575117PMC
September 2008

Evidence for a signaling axis by which intestinal phosphate rapidly modulates renal phosphate reabsorption.

Proc Natl Acad Sci U S A 2007 Jun 12;104(26):11085-90. Epub 2007 Jun 12.

Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA.

The mechanisms by which phosphorus homeostasis is preserved in mammals are not completely understood. We demonstrate the presence of a mechanism by which the intestine detects the presence of increased dietary phosphate and rapidly increases renal phosphate excretion. The mechanism is of physiological relevance because it maintains plasma phosphate concentrations in the normal range after ingestion of a phosphate-containing meal. When inorganic phosphate is infused into the duodenum, there is a rapid increase in the renal fractional excretion of phosphate (FE Pi). The phosphaturic effect of intestinal phosphate is specific for phosphate because administration of sodium chloride does not elicit a similar response. Phosphaturia after intestinal phosphate administration occurs in thyro-parathyroidectomized rats, demonstrating that parathyroid hormone is not essential for this effect. The increase in renal FE Pi in response to the intestinal administration of phosphate occurs without changes in plasma concentrations of phosphate (filtered load), parathyroid hormone, FGF-23, or secreted frizzled related protein-4. Denervation of the kidney does not attenuate phosphaturia elicited after intestinal phosphate administration. Phosphaturia is not elicited when phosphate is instilled in other parts of the gastrointestinal tract such as the stomach. Infusion of homogenates of the duodenal mucosa increases FE Pi, which demonstrates the presence of one or more substances within the intestinal mucosa that directly modulate renal phosphate reabsorption. Our experiments demonstrate the presence of a previously unrecognized phosphate gut-renal axis that rapidly modulates renal phosphate excretion after the intestinal administration of phosphate.
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http://dx.doi.org/10.1073/pnas.0704446104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891094PMC
June 2007

Biological activity of FGF-23 fragments.

Pflugers Arch 2007 Jul 27;454(4):615-23. Epub 2007 Feb 27.

Department of Internal Medicine, Mayo Clinic College of Medicine, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA.

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23 and FGF-23 176-251 significantly and equivalently increased fractional phosphate excretion (FE Pi) from 14 +/- 3 to 32 +/- 5% and 15 +/- 2 to 33 +/- 2% (p < 0.001), respectively. Chronic administration of FGF-23 176-251 reduced serum Pi and serum concentrations of 1alpha,25-dihydroxyvitamin D. Shorter forms of FGF-23 (FGF-23 180-251 and FGF-23 184-251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206-251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180-205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf-23 ( -/- ) knockout mice. The activity of FGF-23 180-251 was confirmed in opossum kidney cells in which the peptide reduced Na(+)-dependent Pi uptake and enhanced internalization of the Na(+)-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic and that a short, 26-amino acid fragment of FGF-23 retains significant phosphaturic activity.
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http://dx.doi.org/10.1007/s00424-007-0231-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818792PMC
July 2007

Biophysical properties of the extra-cellular domain of the calcium-sensing receptor.

Biochem Biophys Res Commun 2006 Oct 17;349(1):339-44. Epub 2006 Aug 17.

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.

The Calcium-Sensing Receptor (CaSR) is a G-protein-coupled receptor that regulates calcium homeostasis by altering parathyroid hormone release, and which binds divalent and trivalent cations, amino acids, polyamines, and polycationic ligands. To obtain information about the structural properties of the CaSR, we expressed milligram quantities of a pure, homogeneous, and functional fragment of the human CaSR extracellular domain (residues 20-535). The expressed and purified protein is folded and binds both neomycin and calcium. It forms dimers in the absence of reducing agents such as beta-mercaptoethanol. Thermal denaturation studies show it has enthalpy and entropy values of unfolding equal to DeltaH=-178+/-4 kJ/mol and DeltaS=-535+/-13 J/mol/K. The protein has significant secondary structure with alpha-helical, beta-sheet, beta-turns, and disordered content of 36.6+/-6.7%, 13.3+/-5.3%, 20.2+/-3.3%, and 29.4+/-4.0%, respectively. The described method for the expression and purification of CaSR should prove useful for further structural studies of this physiologically important protein.
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http://dx.doi.org/10.1016/j.bbrc.2006.08.047DOI Listing
October 2006

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy.

J Am Soc Mass Spectrom 2006 Aug 5;17(8):1158-71. Epub 2006 Jun 5.

Department of Medicine, Mayo Clinic College of Medicine and Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (muESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. Delta 25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary structure.
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http://dx.doi.org/10.1016/j.jasms.2006.04.029DOI Listing
August 2006

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

Pflugers Arch 2006 Jan 9;451(4):579-87. Epub 2005 Sep 9.

Division of Nephrology and Hypertension, Departments of Medicine and Biochemistry and Molecular Biology, Mayo Clinic Rochester, Mayo College of Medicine, MN, 55905, USA.

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.
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http://dx.doi.org/10.1007/s00424-005-1495-2DOI Listing
January 2006

Changes in structure and stability of calbindin-D(28K) upon calcium binding.

Anal Biochem 2004 Nov;334(1):97-105

Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.
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http://dx.doi.org/10.1016/j.ab.2004.07.032DOI Listing
November 2004

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

J Clin Invest 2003 Sep;112(5):785-94

Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.

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.
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http://dx.doi.org/10.1172/JCI18563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC182208PMC
September 2003

The effects of Ca(2+) binding on the conformation of calbindin D(28K): a nuclear magnetic resonance and microelectrospray mass spectrometry study.

Anal Biochem 2003 Jun;317(1):59-66

Duke University NMR Center, Duke University Medical Center, Durham, North Carolina 27710, USA.

Calbindin D(28K) is a six-EF-hand calcium-binding protein found in the brain, peripheral nervous system, kidney, and intestine. There is a paucity of information on the effects of calcium binding on calbindin D(28K) structure. To further examine the mechanism and structural consequences of calcium binding to calbindin D(28K) we performed detailed complementary heteronuclear NMR and microelectrospray mass spectrometry investigations of the calcium-induced conformational changes of calbindin D(28K). The combined use of these two powerful analytical techniques clearly and very rapidly demonstrates the following: (i). apo-calbindin D(28K) has an ordered structure which changes to a notably different ordered conformation upon Ca(2+) loading, (ii). calcium binding is a sequential process and not a simultaneous event, and (iii). EF-hands 1, 3, 4, and 5 take up Ca(2+), whereas EF-hands 2 and 6 do not. Our results support the opinion that calbindin D(28K) has characteristics of both a calcium sensor and a buffer.
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http://dx.doi.org/10.1016/s0003-2697(03)00084-8DOI Listing
June 2003
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