Pit-1 Expression Research Articles

Somatotropinomas, but not nonfunctioning pituitary adenomas, maintain a functional apoptotic RET/Pit1/ARF/p53 pathway that is blocked by excess GDNF.

Acromegaly is caused by somatotroph cell adenomas (somatotropinomas [ACROs]), which secrete GH. Human and rodent somatotroph cells express the RET receptor. In rodents, when normal somatotrophs are deprived of the RET ligand, GDNF (Glial Cell Derived Neurotrophic Factor), RET is processed intracellularly to induce overexpression of Pit1 [Transcription factor (gene : POUF1) essential for transcription of Pituitary hormones GH, PRL and TSHb], which in turn leads to p19Arf/p53-dependent apoptosis. Our purpose was to ascertain whether human ACROs maintain the RET/Pit1/p14ARF/p53/apoptosis pathway, relative to nonfunctioning pituitary adenomas (NFPAs). Apoptosis in the absence and presence of GDNF was studied in primary cultures of 8 ACROs and 3 NFPAs. Parallel protein extracts were analyzed for expression of RET, Pit1, p19Arf, p53, and phospho-Akt. When GDNF deprived, ACRO cells, but not NFPAs, presented marked level of apoptosis that was prevented in the presence of GDNF. Apoptosis was accompanied by RET processing, Pit1 accumulation, and p14ARF and p53 induction. GDNF prevented all these effects via activation of phospho-AKT. Overexpression of human Pit1 (hPit1) directly induced p19Arf/p53 and apoptosis in a pituitary cell line. Using in silico studies, 2 CCAAT/enhancer binding protein alpha (cEBPα) consensus-binding sites were found to be 100% conserved in mouse, rat, and hPit1 promoters. Deletion of 1 cEBPα site prevented the RET-induced increase in hPit1 promoter expression. TaqMan qRT-PCR (real time RT-PCR) for RET, Pit1, Arf, TP53, GDNF, steroidogenic factor 1, and GH was performed in RNA from whole ACRO and NFPA tumors. ACRO but not NFPA adenomas express RET and Pit1. GDNF expression in the tumors was positively correlated with RET and negatively correlated with p53. In conclusion, ACROs maintain an active RET/Pit1/p14Arf/p53/apoptosis pathway that is inhibited by GDNF. Disruption of GDNF's survival function might constitute a new therapeutic route in acromegaly.

High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24h and had shifted more cells from G0/G1 to S+G2/M within 12h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24h after seeding.

Naming an Innominate: Pelvis and Hindlimbs of Miocene Whales Give an Insight into Evolution and Homology of Cetacean Pelvic Girdle

Cetaceans have highly transformed pelvic and hindlimb bones as a secondary adaptation to an aquatic lifestyle. Paleontological records of these bones are very scarce, which hampers interpretations of their homology and evolution. Newly found innominates, femora and tibia of Miocene baleen whales of family Cetotheriidae have primitive structure, which makes it possible to compare them with archaeocetes. As a result, a traditional view of a cetacean innominate as containing vestiges of all three pelvic bones and acetabulum is corroborated by new data and interpretations. A part of ischium (possibly, the superior ramus) is reduced in modern whales, but its vestige can sometimes be observed. A developmental mechanism underlying this pattern can hypothetically involve alterations in Pbx1 and Pbx2, Prrx1 and Prrx2, Pit1 or BMP7 expression: the observed anatomy is similar to reported cases of BMP7 mutations, which are combined with previously reported alterations in Shh regulation. Thus, pelvic and hindlimb reduction in modern cetaceans can in some aspects be compared to ‘Mermaid Syndrome’.

Influence of dietary phosphorus in the regulation of Sodium phosphate cotransporters in uremic rats (LB717)

Hyperphosphatemia is a common and serious complication of chronic kidney disease, contributing to the increased cardiovascular mortality seen in this patient group. The sodium phosphate (P) cotransporters system is involved in intestinal P absorption and is regulated by several factors. We investigate the effect of diets with distinct concentrations of P in the regulation of cotransporters NaPIIb and PiT1 in the intestine of rats. Male Wistar rats (n=66) were used, 33 of which were submitted to nephrectomy 5/6 (Nx) and the others were used as controls (C). The animals received a standard (ST) diet (20% protein and 0.54% P) for 30 days, after they were divided into 2 sets of 3 groups in accordance with the concentration of P in the diet (0.2%, 0.54% or 0.9%) and fed for 2 days. Blood samples, 24h urine, and intestine (duodenum, ileum and jejunum) were accordingly collected. Immunofluorescence, Western blot or ELISA were used to analyze the expressions of the cotransporters and qRT-PCR to evaluate the gene expression of NaPIIb and PiT1. The concentrations of Cr, Pi, FGF23, PTH and Albu were higher in Nx animals. Pu decreased in Nx animals and some of these parameters were modified by the concentration of dietary P. There were significant differences in the expression of PiT1 in jejunum and ileum of Nx animals in diet 0.2% P relative to their respective controls, normalized to B-actin (p=0.01 and p=0.02, respectively). The results demonstrate alteration in P homeostasis in Nx animals impaired upregulation of cotransporters in the P low diet and downregulation in the P high diet differently of the controls, as observed in PCR and immunofluorescence for PiT and NaPi in duodenum, respectively. Grant Funding Source: Supported by FAPESP

The dynamic chromatin configuration during pituitary differentiation (562.1)

Pit‐1, a pituitary specific POU homeodomain transcription factor, plays an essential role in the process of differentiation of anterior pituitary. Abolishing the expression of Pit‐1 results in the combined block in the differentiation of somatotropes, lactotropes, and thyrotropes in anterior pituitary. Prior studies have revealed that the initial activation of Pit‐1 at embryonic day 14 (E14) in the developing mouse pituitary. This activation is mediated by the binding of transcription factor Atbf1 at the ‘early enhancer’ of Pit‐1, located ‐5.8 kb upstream of the Pit‐1 promoter, and by concurrent binding of Prop‐1 at the promoter. The maintenance phase of Pit‐1 expression is initiated at E16.5 by an auto‐regulatory mechanism in which Pit‐1 binds to its own promoter and to a distal enhancer (‘late enhancer’) located 10 kb upstream of the Pit‐1 promoter. Despite the identification of these regulatory elements, the accompanying changes in chromatin architecture at the Pit‐1 locus during the pituitary development remains undefined. In the current study, we investigate the chromatin configuration of the Pit‐1 locus at different developmental stages. We detect acetylation that is restricted to the early enhancer and the promoter at E14.5 while acetylation spreads throughout the entire locus at the adult stage. Our 3C assays reveal that the early enhancer is positioned proximal to the Pit‐1 promoter while in the maintenance phase is accompanied by a switch in the locus conformation, with positioning of the distal enhancer in proximity to the promoter. Furthermore, our studies suggest that the interaction between the distal enhancer and the promoter is mediated by Pit‐1 and CBP in differentiated pituitary. Taken together, our studies demonstrate that the regulation of Pit‐1 expression is accompanied by the dynamic alternation of the chromatin conformation during the pituitary differentiation and the chromatin architectures play important roles in activation and maintenance of gene expression.Grant Funding Source: Supported by University Research foundation of University of Pennsylvania

Constitutive Somatostatin Receptor Subtype-3 Signaling Suppresses Growth Hormone Synthesis

Somatostatin signals through somatostatin receptor subtypes (SSTR) 2 and 5 to attenuate GH secretion. Although expressed in normal pituitary glands and in GH-secreting pituitary tumors, SSTR3 function was unclear, and we have now determined the role of SSTR3 in somatotroph function. Stable rat pituitary tumor cell (GC) transfectants of human SSTR3 (GpSSTR3WT) showed suppression of rat (r) GH promoter activity, GH mRNA expression, and secreted GH concordant with suppressed cAMP/protein kinase A (PKA) signaling. In contrast, cAMP levels and GH expression were unchanged in cells expressing a mutant SSTR3 DRY motif (GpSSTR3R141A). GH expression was rescued by treatment of GpSSTR3WT with forskolin and 8-bromo-cAMP. GpSSTR3WT exhibited activation of glycogen synthase kinase3-β (GSK3-β), a PKA substrate, which was also reversed by 8-Bromo-cAMP treatment. Moreover, SSTR3-dependent GH transcriptional inhibition was rescued by inhibition of GSK3-β. GpSSTR3WT exhibited elevated Pit-1 serine phosphorylation and decreased Pit-1 occupancy of the rGH promoter with sustained Pit-1 expression. GSK3-β and Pit-1 physically interacted with each other, indicating that Pit-1 may be a GSK3-β phosphorylation substrate. In conclusion, constitutive SSTR3 activity mediates transcriptional repression of GH through cAMP/PKA, leading to subsequent activation of GSK3-β and increased Pit-1 phosphorylation and ultimately attenuating Pit-1 binding to the rGH promoter.

Expression of Osteogenic Molecules in the Caudate Nucleus and Gray Matter and Their Potential Relevance for Basal Ganglia Calcification in Hypoparathyroidism

Basal ganglia calcification (BGC) is an interesting example of ectopic calcification in patients with hypoparathyroidism. Its pathogenesis and reasons for predilection of calcification at basal ganglia are not clear. To assess the expression of osteogenesis-related molecules in the caudate nucleus and surface gray matter (an area spared from calcification) and discuss potential relevance of the results in context of BGC in idiopathic hypoparathyroidism. Caudate nucleus and gray matter were obtained from 14 autopsies performed in accidental deaths. The mRNA expression of bone transcription factors (RUNX2/osterix), bone morphogenetic proteins (BMPs) 2 and 4, osteonectin, osteopontin, osteocalcin, vitamin D receptor, calcium sensing-receptor, Na phosphate transporters (PiTs) 1 and 2, N-methyl-D-aspartate receptor 2B (NMDAR2B), carbonic anhydrase II (CA-II), PTH1 receptor (PTH1R), PTH2R, and PTHrP were assessed by RT-PCR. Western blot, spot densitometry, and immunohistochemistry were performed to assess protein expression of molecules showing differences in mRNA expression between caudate and gray tissues. The mean mRNA expression of PiT1 (11.0 ± 10.39 vs 32.9 ± 20.98, P = .003) and PTH2R (1.6 ± 1.47 vs 13.7 ± 6.11, P = .001) were significantly lower in the caudate nucleus than the gray matter. The expression of osteonectin, osteopontin, and CA-II were significantly higher in the caudate nucleus than the gray matter (P = .01, .001, and .04, respectively). The mRNA expression of other molecules was comparable in the 2 tissues. The protein expression of both CA-II and osteonectin was 24% higher and PiT1 17% lower in caudate than the gray matter. The differences in the PTH2R and osteopontin protein expression were not appreciable. The presence of several osteogenic molecules in caudate nucleus indicates that BGC would probably be the outcome of an active process. The differences in expression of these molecules in caudate over gray matter could favor BGC at this site in the unique biochemical milieu of hypoparathyroid state.

Adipose Tissue Macrophages (ATM) of obese patients are releasing increased levels of prolactin during an inflammatory challenge: A role for prolactin in diabesity?

BackgroundObesity, characterized by low grade inflammation, induces adipose tissue macrophage (ATM) infiltration in white adipose tissue (AT) in both humans and rodents, thus contributing to insulin resistance. Previous studies have shown altered prolactin secretion in obesity, however, studies linking ATM infiltration and prolactin (PRL) secretion to the pathogenesis of the metabolic syndrome, obesity and diabetes are lacking. Methods/ResultsIn vivo, qPCR and Western blot analysis demonstrated that prolactin expression was increased in AT of obese rats and also in human AT from obese, obese pre-diabetic and obese diabetic compared to lean counterparts. Immunohistochemistry of obese rat and human AT sections demonstrated a specific expression of prolactin in macrophages. In vitro, we demonstrated that hyperglycemia and inflammation stimulated macrophages (human THP-1 cell line and sorted rat ATM) to express PRL, when challenged with different glucose concentrations with or without IL1β. In in vivo and in vitro experiments, we assessed the expression of Pit-1 (PRL-specific transcription factor) and found that its expression was parallel to PRL expression. ConclusionsIn this study, we show that rodent and human macrophages synthesize prolactin in response to inflammation and high glucose concentrations. General significanceOur data shed new light on the potential role of macrophages in the physiopathology of diabesity via the PRL expression and on its expression mechanism and regulation.

MiRNA‐221 and miRNA‐222 synergistically function to promote vascular calcification

Vascular calcification shares many similarities with skeletal mineralisation and involves the phenotypic trans-differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation; however, their role in mediating VSMC calcification is not fully understood. miR-microarray analysis revealed the significant down-regulation of a range of miRs following nine days in culture, including miR-199b, miR-29a, miR-221, miR-222 and miR-31 (p < 0.05). Subsequent studies investigated the specific role of the miR-221/222 family in VSMC calcification. Real-time quantitative polymerase chain reaction data confirmed the down-regulation of miR-221 (32.4%; p < 0.01) and miR-222 (15.7%; p < 0.05). VSMCs were transfected with mimics of miR-221 and miR-222, individually and in combination. Increased calcium deposition was observed in the combined treatment (two-fold; p < 0.05) but not in individual treatments. Runx2 and Msx2 expression was increased during calcification, but no difference in expression was observed following transfection with miR mimics. Interestingly, miR-221 and miR-222 mimics induced significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit-1 expression, suggesting that these miRs may modulate VSMC calcification through cellular inorganic phosphate and pyrophosphate levels. © 2013 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.

Loss of Function of Slc20a2 Associated with Familial Idiopathic Basal Ganglia Calcification in Humans Causes Brain Calcifications in Mice

Familial idiopathic basal ganglia calcification (FIBGC) is a neurodegenerative disorder with neuropsychiatric and motor symptoms. Deleterious mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), were recently linked to FIBGC in almost 50 % of the families reported worldwide. Here, we show that knockout of Slc20a2 in mice causes calcifications in the thalamus, basal ganglia, and cortex, demonstrating that reduced PiT2 expression alone can cause brain calcifications.

Localization of type-III sodium-dependent phosphate transporter 2 in the mouse brain

Type-III sodium-dependent phosphate transporters 1 and 2 (PiT-1 and PiT-2, respectively) are proteins encoded by SLC20A1 and SLC20A2, respectively. The ubiquitous distribution of PiT-1 and PiT-2 mRNAs in mammalian tissues is in agreement with the housekeeping maintenance of homeostasis of intracellular inorganic phosphate (Pi), which is absorbed from interstitial fluid for normal cellular functions. Recently, mutations of SLC20A2 have been found in patients with idiopathic basal ganglia calcification (IBGC), also known as Fahr's disease. However, the localization of PiT-2 in the brain has not been clarified yet. Therefore, the aim of this study is to clarify the distribution of PiT-2 expression in the mouse brain. Our biochemical and immunohistochemical analyses using a polyclonal antibody (Ab) and a monoclonal Ab showed that PiT-2 was ubiquitously expressed throughout the brain. In terms of the cellular type, PiT-2 was predominantly detected in neurons; it colocalized with β-tubulin III in the cerebral cortex and with calbindin D-28K in Purkinje cells. Additionally, PiT-2 immunopositivity was detected in the microtubule-associated protein 2-positive neuronal dendrites in the cerebral cortex. However, colocalization with PiT-2 immunopositivity was not observed in the synaptophysin-positive nerve terminals. PiT-2 was also expressed in astrocytes and vascular endothelial cells. Our results indicate that PiT-2 plays an important role in the maintenance of cellular Pi homeostasis in neurons, astrocytes, and endothelial cells. This finding is a milestone in the study of the function of PiT-2 in the normal mouse brain and particularly in the brains of patients with Fahr's disease.

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

Mice with Hypomorphic Expression of the Sodium-Phosphate Cotransporter PiT1/Slc20a1 Have an Unexpected Normal Bone Mineralization

The formation of hydroxyapatite crystals and their insertion into collagen fibrils of the matrix are essential steps for bone mineralization. As phosphate is a main structural component of apatite crystals, its uptake by skeletal cells is critical and must be controlled by specialized membrane proteins. In mammals, in vitro studies have suggested that the high-affinity sodium-phosphate cotransporter PiT1 could play this role. In vivo, PiT1 expression was detected in hypertrophic chondrocytes of murine metatarsals, but its implication in bone physiology is not yet deciphered. As the complete deletion of PiT1 results in embryonic lethality at E12.5, we took advantage of a mouse model bearing two copies of PiT1 hypomorphic alleles to study the effect of a low expression of PiT1 on bone mineralization in vivo. In this report, we show that a 85% down-regulation of PiT1 in long bones resulted in a slight (6%) but significant reduction of femur length in young mice (15- and 30-day-old). However, despite a defect in alcian blue / alizarin red S and Von Kossa staining of hypomorphic 1-day-old mice, using X-rays micro-computed tomography, energy dispersive X-ray spectroscopy and histological staining techniques we could not detect differences between hypomorphic and wild-type mice of 15- to 300-days old. Interestingly, the expression of PiT2, the paralog of PiT1, was increased 2-fold in bone of PiT1 hypomorphic mice accounting for a normal phosphate uptake in mutant cells. Whether this may contribute to the absence of bone mineralization defects remains to be further deciphered.

Sodium phosphate cotransporters in intestinal absorption of phosphorus in uremic rats

Hyperphosphatemia is a common disorder in patients with chronic kidney disease (CKD). We investigate the effect of diets with distinct concentrations of P in the regulation of cotransporters NaPIIb and PiT1 in the intestine of rats. 66 male Wistar rats were used, 33 of which were submitted to nephrectomy 5/6 (Nx) and the others were used as controls (C). The animals received a standard (ST) diet (20% protein and 0.54% P) for 30 days, after they were divided into 2 sets of 3 groups in accordance with the concentration of P in the diet (0.2%↓, 0.54% or 0.9%↑) and fed for 2 days. Blood samples and 24h urine were collected and the expressions of the cotransporters were analyzed by immunofluorescence and Western blot (WB). The table summarize the results (mean ± SD). NaPIIb in ileum of Nx rats. (A) Negative control (B) ↓P; (C) ST; (D) ↑P The concentrations of Cr, P, FGF23, PTH and Albu were higher in Nx animals. Pu decreased in Nx animals and some of these parameters were modified by the concentration of dietary P. The expression of NaPIIb and PiT1 and the WB analysis are being finalized.

Vascular calcification--is aldosterone a culprit?

In chronic kidney disease (CKD), increased plasma phosphate concentrations cause vascular calcification which substantially contribute to cardiovascular events and increased mortality of CKD patients. Similar to CKD patients, klotho-hypomorphic mice (kl/kl) also suffer from excessive vascular calcification leading to growth deficit, rapid ageing and early death. The hyperphosphataemia of kl/kl mice results from excessive formation of 1,25(OH)2D3 causing excessive intestinal phosphate absorption. Further, kl/kl mice further suffer from hyperaldosteronism and compelling evidence points to an active role of mineralocorticoids in triggering osteoinductive programmes in the vasculature, thus further contributing to the development of vascular calcification. Conversely, in kl/kl mice, the mineralocorticoid receptor antagonist spironolactone decreased the vascular osteoinductive processes and reversed the excessive expression of osteogenic programmes, i.e. type III sodium-dependent phosphate transporter Pit1, tumour necrosis factor α (Tnfα), transcription factors Msx2, Cbfa1/Runx2 and osterix as well as alkaline phosphatase (Alp). In human aortic vascular smooth muscle cells (HAoSMCs), aldosterone alone similarly triggered an 'osteogenic' programme, thus increasing PIT1, TNFα, MSX2, CBFA1/RUNX2 and ALP expression as well as ALP activity and potentiated the effects of phosphate treatment. These effects were again reversed by spironolactone and in addition by PIT1 silencing. The above observations reveal that the severe vascular calcification is not only the result of high plasma phosphate concentrations, but also promoted by aldosterone-driven osteoinductive signalling. Future studies in CKD patients will be required to define the role of aldosterone and the potential impact of its inhibition by spironolactone in the pathophysiology of vascular calcification.

EKLF-driven PIT1 expression is critical for mouse erythroid maturation in vivo and in vitro

AbstractThe PIT1/SLC20A1 protein, a well-described sodium/phosphate cotransporter and retrovirus receptor, has been identified recently as a modular of proliferation and apoptosis in vitro. The targeted deletion of the PIT1 gene in mice revealed a lethal phenotype due to severe anemia attributed to defects in liver development. However, the presence of immature erythroid cells associated with impaired maturation of the globin switch led us to investigate the role of PIT1 in hematopoietic development. In the present study, specific deletion of PIT1 in the hematopoietic system and fetal liver transplantation experiments demonstrated that anemia was associated with an erythroid cell– autonomous defect. Moreover, anemia was not due to RBC destruction but rather to maturation defects. Because Erythroid Krüppel-like Factor (EKLF)–knockout mice showed similar maturation defects, we investigated the functional link between PIT1 and EKLF. We demonstrated that EKLF increases PIT1 expression during RBC maturation by binding to its promoter in vivo and that shRNA-driven depletion of either PIT1 or EKLF impairs erythroid maturation of G1E cells in vitro, whereas reexpression of PIT1 in EKLF-depleted G1E cells partially restores erythroid maturation. This is the first demonstration of a physiologic involvement of PIT1 in erythroid maturation in vivo.

Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice

Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH)2D3] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH)2D3, FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

Ox-LDL Induces Expression of PiT-1 in Human Aortic Valvular Interstitial Cells

Ox-LDL Induces Expression of PiT-1 in Human Aortic Valvular Interstitial Cells

Expression of Steroidogenic Factor 1 and Pituitary Specific Transcription Factor 1 in Rat Pituitary Adenomas

The protein expressions of steroidogenic factor l (SF-l) and pituitary-specific transcription factor 1 (Pit-1) were investigated immunohistochemically for 53 spontaneous pituitary adenomas of the pars distalis from male Crl:CD(SD) rats. Luteinizing hormone (LH)-positive/prolactin (PRL)-negative and LH-negative/PRL-positive adenomas showed that the expression of SF-1 and Pit-1 was exclusively related to the immunoreactivity of LH and PRL, respectively. All double-positive adenomas (positive for both LH and PRL) were positive for Pit-1 and were supposed to be derived from PRL cells, although some of them also showed SF-1 immunoreactivity. In addition, all null cell adenomas (negative for all anterior pituitary hormones) were positive for SF-1 and negative for Pit-1, indicating that they originated from the gonadotroph cell lineage. This is the first report focusing on the application of transcription factors for the classification of rat pituitary adenomas.

NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.