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The polycystin–1 (PC1), polycystin–2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome–like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C–terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C–terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C–terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is > 2 MDa in size and that N–terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.

Background The major form of autosomal dominant polycystic kidney disease is caused by heterozygous mutations in PKD1 , the gene that encodes polycystin-1 (PC1). Unlike PKD1 genes in the mouse and most other mammals, human PKD1 is unusual in that it contains two long polypyrimidine tracts in introns 21 and 22 (2.5 kbp and 602 bp, respectively; 97% cytosine and thymine). Although these polypyrimidine tracts have been shown to form thermodynamically stable segments of triplex DNA that can cause DNA polymerase stalling and enhance the local mutation rate, the efficiency of transcription and splicing across these cytosine- and thymine-rich introns has been unexplored. Methods We used RT-PCR and Western blotting (using an mAb to the N terminus) to probe splicing events over exons 20–24 in the mouse and human PKD1 genes as well as Nanopore sequencing to confirm the presence of multiple splice forms. Results Analysis of PC1 indicates that humans, but not mice, have a smaller than expected protein product, which we call Trunc_PC1. The findings show that Trunc_PC1 is the protein product of abnormal differential splicing across introns 21 and 22 and that 28.8%–61.5% of PKD1 transcripts terminate early. Conclusions The presence of polypyrimidine tracts decreases levels of full-length PKD1 mRNA from normal alleles. In heterozygous individuals, low levels of full-length PC1 may reduce polycystin signaling below a critical “cystogenic” threshold.

Nephrogenic systemic fibrosis (NSF) is a devastating condition associated with gadolinium (Gd3+)-based contrast agents (GBCAs) in patients with kidney disease. The release of toxic Gd3+ from GBCAs likely plays a major role in NSF pathophysiology. The cause and etiology of Gd3+ release from GBCAs is unknown. Increased Acidic Serine Aspartate Rich MEPE-associated peptides (ASARM peptides) induce bone mineralization abnormalities and contribute to renal phosphate-handling defects in inherited hypophosphatemic rickets and tumor-induced osteomalacia. The proteolytic cleavage of related bone matrix proteins with ASARM motifs results in release of ASARM peptide into bone and circulation. ASARM peptides are acidic, reactive, phosphorylated inhibitors of mineralization that bind Ca2+ and hydroxyapatite. Since the ionic radius of Gd3+ is close to that of Ca2+, we hypothesized that ASARM peptides increase the risk of NSF by inducing release of Gd3+ from GBCAs. Here, we show 1) ASARM peptides bind and induce release of Gd3+ from GBCAs in vitro and in vivo; 2) A bioengineered peptide (SPR4) stabilizes the Gd3+-GBCA complex by specifically binding to ASARM peptide in vitro and in vivo; and 3) SPR4 peptide infusion prevents GBCA-induced NSF-like pathology in a murine model with increased ASARM peptide (Hyp mouse). We conclude ASARM peptides may play a role in NSF and SPR4 peptide is a candidate adjuvant for preventing or reducing risk of disease.

Context Mice with null mutations in matrix extracellular phosphoglycoprotein (MEPE) have increased bone mass, increased trabecular density and abnormal cancellous bone (MN-mice). These defects worsen with age and MEPE overexpression induces opposite effects. Also, genome wide association studies show that MEPE plays a major role in bone mass. We hypothesized that the conserved C-terminal MEPE ASARM-motif is chiefly responsible for regulating bone mass and trabecular structure. Design To test our theory we overexpressed C-terminal ASARM-peptide in MN-mice using the Col1α1 promoter (MNAt-mice). We then compared the bone and renal phenotypes of the MNAt-mouse with the MN-mouse and the X-linked hypophosphatemic rickets mouse (HYP). The HYP mouse overexpresses ASARM-peptides and is defective for the PHEX gene. Results The MN-mouse developed increased bone mass, bone strength and trabecular abnormalities that worsened markedly with age. Defects in bone formation were chiefly responsible with suppressed sclerostin and increased active β-catenin. Increased uric acid levels also suggested that abnormalities in purine-metabolism and a reduced fractional excretion of uric acid signaled additional renal transport changes. The MN mouse developed a worsening hyperphosphatemia and reduced FGF23 with age. An increase in the fractional excretion of phosphate (FEP) despite the hyperphosphatemia confirms an imbalance in kidney–intestinal phosphate regulation. Also, the MN mice showed an increased creatinine clearance suggesting hyperfiltration. A reversal of the MN bone–renal phenotype changes occurred with the MNAt mice including the apparent hyperfiltration. The MNAt mice also developed localized hypomineralization, hypophosphatemia and increased FGF23. Conclusions The C-terminal ASARM-motif plays a major role in regulating bone-mass and cancellous structure as mice age. In healthy mice, the processing and release of free ASARM-peptide are chiefly responsible for preserving normal bone and renal function. Free ASARM-peptide also affects renal mineral phosphate handling by influencing FGF23 expression. These findings have implications for understanding age-dependent osteoporosis, unraveling drug-targets and developing treatments.

Context ASARM-peptides are substrates and ligands for PHEX, the gene responsible for X-linked hypophosphatemic rickets (HYP). PHEX binds to the DMP1-ASARM-motif to form a trimeric-complex with α5β3-integrin on the osteocyte surface and this suppresses FGF23 expression. ASARM-peptide disruption of this complex increases FGF23 expression. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide and ASARM-motif to study DMP1–PHEX interactions and to assess SPR4 for treating inherited hypophosphatemic rickets. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle into wild-type mice (WT) and HYP-mice for 4 weeks. Results Asymmetrically distributed mineralization defects occurred with WT-SPR4 femurs. Specifically, SPR4 induced negative effects on trabecular bone and increased bone volume and mineralization in cortical-bone. Markedly increased sclerostin and reduced active β-catenin occurred with HYP mice. SPR4-infusion suppressed sclerostin and increased active β-catenin in WT and HYP mice and improved HYP-mice trabecular mineralization defects but not cortical mineralization defects. Conclusions SPR4-peptide has bimodal activity and acts by: (1) preventing DMP1 binding to PHEX and (2) sequestering an inhibitor of DMP1–PHEX binding, ASARM-peptide. In PHEX defective HYP-mice the second pathway predominates. Although SPR4-peptide improved trabecular calcification defects, decreased sclerostin and increased active β-catenin it did not correct HYP-mice cortical mineralization defects on a normal phosphate diet. Thus, for inherited hypophosphatemic rickets patients on a normal phosphate diet, SPR4-peptide is not a useful therapeutic.

Matrix extracellular phosphoglycoprotein (MEPE) belongs to the SIBLING protein family which play key roles in biomineralization. Although the growth plates of MEPE-overexpressing mice display severe morphological disruption, the expression and function of MEPE in growth plate matrix mineralization remains largely undefined. Here we show MEPE and its cleavage product, the acidic serine aspartate-rich MEPE-associated motif (ASARM) peptide, to be localised to the hypertrophic zone of the growth plate. We also demonstrate that the phosphorylated (p)ASARM peptide inhibits ATDC5 chondrocyte matrix mineralization. Stable MEPE-overexpressing ATDC5 cells also had significantly reduced matrix mineralization in comparison to the control cells. Interestingly, we show that the addition of the non-phosphorylated (np)ASARM peptide promoted mineralization in the ATDC5 cells. The peptides and the overexpression of MEPE did not affect the differentiation of the ATDC5 cells. For a more physiologically relevant model, we utilized the metatarsal organ culture model. We show the pASARM peptide to inhibit mineralization at two stages of development, as shown by histological and μCT analysis. Like in the ATDC5 cells, the peptides did not affect the differentiation of the metatarsals indicating that the effects seen on mineralization are direct, as is additionally confirmed by no change in alkaline phosphatase activity or mRNA expression. In the metatarsal organ cultures, the pASARM peptide also reduced endothelial cell markers and vascular endothelial growth factor mRNA expression. Taken together these results show MEPE to be an important regulator of growth plate chondrocyte matrix mineralization through its cleavage to an ASARM peptide., Highlights ► MEPE and its ASARM peptide are localised to the hypertrophic chondrocytes of the growth plate. ► Phosphorylated ASARM peptides inhibit chondrocyte matrix mineralisation in ATDC5 cells and in metatarsal organ cultures. ► This inhibition is independent of altered alkaline phosphatase activity. ► Phosphorylated ASARM peptides inhibited the mRNA expression of endothelial cell specific markers and pro-angiogenic VEGF.

Transgenic mice that express Cre recombinase in erythroid cell lineages were developed so that genes affecting erythropoiesis/hematopoiesis may be altered without necessarily affecting fetus viability. A micro-LCR cassette-beta-globin promoter-Cre recombinase gene (microLCR-betapr-Cre) construct was synthesized and used to generate transgenic mice. Concurrently, we produced mice containing a microLCR-loxP-flanked beta sickle gene (microLCR-loxP-beta(S)-loxP) construct. microLCR-betapr-Cre mice with intact transgenes in variable copy number were identified. Cre expression was assessed by RNAse protection and RT-PCR. Cre function was ascertained by breeding to microLCR-loxP-beta(S)-loxP mice. We demonstrate that beta(S) expression was not detected in the blood of bigenics, but the gene was present in nonerythroid cells. Thus, excision of the loxP-flanked beta(S) gene was restricted to erythroid cell lineages.

The identity of the cell type responsive to sclerostin, a negative regulator of bone mass, is unknown. Since sclerostin is expressed in vivo by mineral-embedded osteocytes, we tested the hypothesis that sclerostin would regulate the behavior of cells actively involved in mineralization in adult bone, the preosteocyte. Differentiating cultures of human primary osteoblasts exposed to recombinant human sclerostin (rhSCL) for 35 days displayed dose- and time-dependent inhibition of in vitro mineralization, with late cultures being most responsive in terms of mineralization and gene expression. Treatment of advanced (day 35) cultures with rhSCL markedly increased the expression of the preosteocyte marker E11 and decreased the expression of mature markers DMP1 and SOST. Concomitantly, matrix extracellular phosphoglycoprotein (MEPE) expression was increased by rhSCL at both the mRNA and protein levels, whereas PHEX was decreased, implying regulation through the MEPE-ASARM axis. We confirmed that mineralization by human osteoblasts is exquisitely sensitive to the triphosphorylated ASARM-PO4 peptide. Immunostaining revealed that rhSCL increased the endogenous levels of MEPE-ASARM. Importantly, antibody-mediated neutralization of endogenous MEPE-ASARM antagonized the effect of rhSCL on mineralization, as did the PHEX synthetic peptide SPR4. Finally, we found elevated Sost mRNA expression in the long bones of HYP mice, suggesting that sclerostin may drive the increased MEPE-ASARM levels and mineralization defect in this genotype. Our results suggest that sclerostin acts through regulation of the PHEX/MEPE axis at the preosteocyte stage and serves as a master regulator of physiologic bone mineralization, consistent with its localization in vivo and its established role in the inhibition of bone formation.

ContextPHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders.DesignSubcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks.ResultsSPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice.ConclusionsASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of sclerostin and/or sequestration of ASARM-peptides improves energy metabolism and may have utility for treating familial rickets, osteoporosis, obesity and diabetes.

Decades of research has established that the most effective treatment for sickle cell disease (SCD) and Cooley’s anemia is increased fetal hemoglobin (HbF). Certain β-thalassemias may also benefit from fetal hemoglobin induction. Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the most widely available treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Identification of a drug specific for inducing or reactivating γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. An assay system developed in our lab to understand the mechanisms underlying the γ- to β-globin gene expression switch during development allowed us to generate a cell-based assay that was adapted for a HTS of 121,085 compounds from the libraries of the KU-HTS Laboratory (Prestwick, MicroSource, CMLD, Chembridge and ChemDiv compound libraries) and LCGC (OCL compound library). Transgenic mice were produced using a modified 213 Kb human β-globin locus yeast artificial chromosome (β-YAC). Two gene fusions were introduced into the β-YAC via homologous recombination in the host yeast, firefly luciferase was fused to the Aγ-globin promoter and Renilla luciferase was fused to the β-globin promoter. The resultant YAC was microinjected into fertilized mouse oocytes to produce transgenic mice. We used these mice to derive chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) containing the γ-luc/β-luc β-YAC, which were employed in the HTS. We identified 232 primary screen actives that induced γ-globin 2-fold or higher. A 4-assay, 10-point dose-response secondary screen using the same CID-dependent γ-luc/β-luc β-YAC BMCs reconfirmed that 211 of these active compounds induced γ-globin ≥2-fold with minimal or no β-globin induction, minimal cytotoxicity and did not directly inhibit purified luciferase enzyme. Additional secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the characteristics of seven of these 233 hits that were cherry-picked for further analysis. Four of the compounds were particularly promising, numbers 7, 42, 87 and 208. In CID-dependent wild-type β-YAC BMCs using the optimal dose for each compound, γ-globin mRNA induction ranged from 3- to 42-fold compared to 10-fold with sodium butyrate as measured by real-time qRT-PCR; F-cells ranged from 9.9-29.9% compared to 0.7% untreated and 15.9% treated with sodium butyrate as measured by flow cytometry. In human primary erythroid progenitor cells, the mRNA change was 1.6- to 3-fold compared to 1.75-fold with sodium butyrate and F-cells ranged from 9.1-29% compared to 5.7% untreated and 39.4% treated with sodium butyrate. Lead compounds will be tested in a pre-clinical β-YAC transgenic mouse model to determine their ability to induce HbF in vivo to aid development of these compounds for future clinical applications in hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

Abstract 353 Sickle cell disease (SCD) impacts one of 400 African-Americans born each year. Augmentation of fetal hemoglobin (HbF) levels is widely accepted as the most effective method for treating SCD, but hydroxyurea (HU) is currently the only approved drug that increases HbF. Thus, there is a need for the development of new therapies for this disease, including the identification of transcriptional activators that specifically up-regulate γ-globin (HbF). Developmental regulation of human β-like globin gene switching is controlled by several parameters, including cis- and trans-acting transcriptional determinants. Understanding the mechanisms underlying control of globin gene expression, particularly those involved in activation of γ-globin expression (HbF) is important for developing new treatments for SCD. Metal-responsive transcription factor-1 (MTF-1) is a key regulator of zinc metabolism in higher eukaryotes that controls the metal-inducible expression of metallothioneins and a number of other genes directly involved in the intracellular sequestration and efflux transport of zinc. Previous studies demonstrated that MTF-1 plays an essential role in liver development and that MTF-1-deficient mice display an anemic phenotype, suggesting a role for MTF-1 in hematopoiesis. In our study, when murine MTF-1 was expression was enforced, we observed a 5-fold increase in γ-globin expression in K562 cells. We also demonstrated increased γ-globin expression in adult blood from MTF-1 human β-globin locus yeast artificial chromosome (β-YAC) bi-transgenic (bigenic) mouse lines at the mRNA level by quantitative real-time RT-PCR (qPCR) and at the protein level by FACS analysis. Lastly, γ-globin gene expression was induced 12-fold in bone marrow cells (BMCs) derived from these bigenic mice compared to BMCs derived from β-YAC-only mice, and 3-fold after 6 hours of zinc treatment in β-YAC-only BMCs. Corroborative studies including zinc-deficient and zinc replete diets in β-YAC mice and erythroid-specific MTF-1 loss-of-function in loxP-flanked-MTF-1 LCR-β-globin promoter-Cre β-YAC mice further support a role for MTF-1 in g-globin gene expression. Chromatin immunoprecipitation (ChIP) analysis did not show recruitment of MTF-1 to any γ-globin gene-proximal metal response elements (MREs), the DNA motif that MTF-1 binds to control zinc metabolism genes. However, GATA-2 co-immunoprecipitated with MTF-1 in MTF-1 β-YAC BMCs, but not in β-YAC-only BMCs, suggesting that reactivation of γ-globin expression by MTF-1 might be mediated by a MTF-1-GATA-2 protein complex. ChIP experiments indicated that MTF-1 and GATA-2 co-occupy the same sites in the γ-globin promoter. Two of the stronger co-recruitment regions contain not only GATA sites, but also non-canonical MREs that vary by 1 or 2 bp from the canonical 7 bp MRE core. Interestingly, GATA-2 was induced 2-fold in adult blood of MTF-1 β-YAC mice, and also 3.5-fold in MTF-1 β-YAC BMCs treated with zinc for 6 hours. Our data suggest that activation of γ-globin by MTF-1 is mediated by protein-protein interaction with GATA-2 and that this multi-protein complex is targeted to GATA sites located in the γ-globin gene-promoters via binding of the GATA-2 protein. In a previous study we identified testis-specific protein Y-like 1 (TSPYL1) as a candidate gene involved in activation of γ-globin (de Andrade et al., 2006, Blood Cells, Mol. & Dis. 37:82). TSPYL1 mRNA level was increased 2–5 fold in deletional hereditary persistence of fetal hemoglobin (HPFH-2) subjects and decreased in a carrier of the Sicilian δβ-thalassemia trait. TSPYL1 is a transcription factor that is a member of the nucleosome assembly protein (NAP) family. TSPYL1 is not a DNA-binding protein; thus it exerts its effect through protein-protein interactions. When we enforced expression of human TSPYL1 in K562 cells an 11-fold induction of γ-globin expression was obtained. A reduction of γ-globin expression was observed following TSPYL1 knockdown in K562 cells. qPCR analysis of blood from TSPYL1 β-YAC bigenic mice showed that γ-globin expression was increased 4–12-fold. Taken together, our data strongly support the evidence that MTF-1 and TSPYL1 reactivate γ-globin expression in adult erythropoiesis. These two proteins represent potential new targets in strategies to reactivate γ-globin in hemoglobinopathies where higher levels of HbF would have beneficial effects. Disclosures: No relevant conflicts of interest to declare.