Search

Ectopic calcification of soft tissues can have severe clinical consequences especially when localized to vital organs such as heart, arteries and kidneys. Mammalian stanniocalcin (STC) 1 and 2 are glycoprotein hormones identified as calcium/phosphate-regulating hormones. The mRNA of STCs is upregulated in the kidney of α-klotho mutant (kl/kl) mice, which have hypercalcemia, hyperphosphatemia and hypervitaminosis D and exhibit a short life span, osteopenia and ectopic calcification. In the present study, we investigated the distribution and localization of STCs in kl/kl mice. Quantitative RT-PCR revealed that renal mRNA expression of STC2 was increased in both kl/kl mice and fibroblast growth factor 23 (Fgf23)-null mice compared with wild type mice. Interestingly, STC2 protein was focally localized with the calcified lesions of renal arterioles, renal tubular cells, heart and aorta in kl/kl mice. In vitro analysis of rat aortic vascular smooth muscle (A-10) cells showed that inorganic phosphate (Pi) stimulation significantly increased STC2 mRNA levels as well as that of osteocalcin, osteopontin and the type III sodium-dependent phosphate co-transporter (PiT-1), and induced STC2 secretion. Interestingly, the knockdown with a small interfering RNA or the over-expression of STC2 showed acceleration and inhibition of Pi-induced calcification in A-10 cells, respectively. These results suggest that the up-regulation of STC2 gene expression resulting from abnormal α-klotho-Fgf23 signaling may contribute to limitation of ectopic calcification and thus STC2 represents a novel target gene for cardio-renal syndrome.

Stanniocalcin 1 and stanniocalcin 2 are two glycoprotein hormones, which act as calcium phosphate-regulating factor on intestine and kidney. We have previously reported that stanniocalcin 2 expression is positively and negatively controlled by 1,25(OH)(2)D(3) and parathyroid hormone in renal proximal tubular cells. However, it has been unclear whether they regulate the stanniocalcin 1 gene expression. In this study, we identified the opossum stanniocalcin 1 cDNA sequence. The opossum stanniocalcin 1 amino acid sequence had 83% homology with human stanniocalcin 1, and has a conserved putative N-linked glycosylation site. Real-time PCR analysis using opossum kidney proximal tubular (OK-P) cells revealed that the mRNA levels of stanniocalcin 1 gene is up-regulated by both 1,25(OH)(2)D(3) and parathyroid hormone in dose-dependent and time-dependent manners. We also demonstrated that the stanniocalcin 1 expression was increased in parathyroid hormone injected rat kidney. Furthermore, the mRNA expression of stanniocalcin 1 and stanniocalcin 2 were oppositely regulated by phorbol 12,13-myristic acetate, a specific PKC activator. Interestingly, the up-regulation of stanniocalcin 1 gene by 1,25(OH)(2)D(3) and phorbol 12,13-myristic acetate were not prevented in the presence of actinomycin D, an RNA synthesis inhibitor. These results suggest that the stanniocalcin 1 gene expression is up-regulated by 1,25(OH)(2)D(3) and parathyroid hormone through mRNA stabilization in renal proximal tubular cells.

The primary determinant of circulating 1α,25-dihydroxyvitamin D (1,25[OH](2)D) levels is the activity of 25-hydroxyvitamin D-1α-hydroxylase (cytochrome P450 27B1 [CYP27B1]) in the kidney. Hyperthyroid patients have been reported to have low levels of plasma 1,25(OH)(2)D. However, the detailed mechanism of thyroid hormone action on vitamin D metabolism is still poorly understood. The present study determined whether renal CYP27B1 gene expression was negatively regulated by thyroid hormones. T(3)-induced hyperthyroid mice showed marked decreases in plasma 1,25(OH)(2)D levels and in renal expression of CYP27B1 mRNA but no changes in plasma concentrations of calcium, PTH, or fibroblast growth factor-23. In addition, we observed that T(3) administration significantly decreased plasma 1,25(OH)(2)D and renal CYP27B1 mRNA levels that were increased by low-calcium or low-phosphorus diets and induced hypocalcemia in mice fed a low-calcium diet. Promoter analysis revealed that T(3) decreases the basal transcriptional activity of the CYP27B1 gene through thyroid hormone receptors (TRα and TRβ1) and the retinoid X receptor α (RXRα) in renal proximal tubular cells. Interestingly, we identified an everted repeat negative thyroid hormone response element (1α-nTRE) overlapping the sterol regulatory element (1α-SRE) and the TATA-box -50 to -20 base pairs from the human CYP27B1 gene transcription start site. Finally, we established that CYP27B1 gene transcription is positively regulated by SRE-binding proteins and that a T(3)-bound TRβ1/RXRα heterodimer inhibits SRE-binding protein-1c-induced transcriptional activity through the 1α-nTRE. These results suggest that transcriptional repression of the CYP27B1 gene by T(3)-bound TRs/RXRα, acting through the 1α-nTRE, results in decreased renal CYP27B1 expression and plasma 1,25(OH)(2)D levels.

Blood platelets can be obtained only by blood donation and reveal short-shelf life by the reason that they must be maintained with plasma at 20-24 degrees with shaking. These factors lead to shortage of donor platelets for clinical use. To overcome this issue, we developed a clinically applicable strategy for the derivation of functional platelets from human pluripotent stem cells (PSCs). We previously reported in vitro culture methods for producing functional platelets from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) (Takayama et al. Blood 2008, J Exp Med 2010). We also have established immortalized megakaryocyte progenitor cell lines (imMKCLs) with long-term expansion capability from hiPSC-derived hematopoietic progenitors with three defined factors, c-MYC, BMI1 and, BCL-XL (Nakamura et al. Cell Stem Cell, 2014). Although imMKCLs can be promising source of functional platelets for transfusion, further inventive efforts are needed to expand imMKCLs more efficiently towards clinical application. Thrombopoietin (TPO) is a cytokine initially identified as the primary regulator of megakaryocyte differentiation and platelet production. TPO is also an essential supplement for expanding platelets from imMKCLs. Recently, several nonpeptidyl small-molecule compounds have been developed to activate the TPO receptor, c-MPL and promote platelet production such as SB-497115 (Eltrombopag), an orally available drug for thrombocytopenia. Chemically synthesized c-MPL agonists reveal the advantage in terms of biological safety, low-immunogenicity or, low-cost manufacturing as compared to peptide-based ligands for platelet production. To obtain a c-MPL agonist that expands imMKCLs more efficiently and cost effectively than recombinant human TPO (rhTPO), we firstly screened small-molecule c-MPL agonists by evaluating its effects on platelet production from hiPSCs. Consequently, we identified “MK-001”, as the most potent compound that increases platelet productivity, as evidenced by the effects of MK-001 on the proliferation, differentiation, cell signaling, and platelet production from imMKCLs. We also studied the functionality of imMKCL-derived platelets. imMKCLs were cultured for 15 days with passage every 3 or 4 days with rhTPO in the presence of either rhTPO, Eltrombopag or MK-001 employing the same method as previously described (Nakamura et al. Cell Stem Cell, 2014). Total cell number was measured by Trypan Blue staining and automated cell counter. On day11, the number of total cells cultured with 200ng/mL of MK-001 was increased >1.5-fold compared with that of 50ng/mL of rhTPO (p2-fold compared with that of rhTPO (p In conclusion, the c-MPL agonist MK-001 could be applicable as an indispensable tool for expansion of platelets from hiPSCs with a combination of imMKCL system. Disclosures Nakauchi: Nissan Chemical Industries: Research Funding. Eto:Nissan Chemical Industries: Research Funding.