Reevaluation of erythropoietin production by the nephron.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in ß-intercalated or non α/non ß-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Vasopressin V1a receptor is required for nucleocytoplasmic transport of mineralocorticoid receptor.

We previously reported that a deficiency in the vasopressin V1a receptor (V1aR) results in type 4 renal tubular acidosis, which suggests that vasopressin exerts direct effects on the physiological actions of aldosterone. We investigated the role of vasopressin for nucleocytoplasmic transport of mineralocorticoid receptor (MR) in the intercalated cells. Vasopressin V1aR-deficient (V1aR(-/-)) mice showed largely decreased expression of MR and 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) in the medulla of the kidney, which was partially ameliorated by fludrocortisone treatment. The incubation of IN-IC cells, an intercalated cell line established from temperature-sensitive SV40 large T antigen-expressing rats, with aldosterone or vasopressin increased the nuclear-to-cytoplasmic ratio of the MR from 11.2 to 47.2% and from 18.7 to 61.2%, respectively, in 30 min without any changes in MR expression from the whole cell extract. The immunohistochemistry analysis of the IN-IC cells revealed the nuclear accumulation of MRs after a 30-min incubation with aldosterone or vasopressin. These effects were accompanied by an increase in regulator of chromosome condensation-1 (RCC-1) due to aldosterone and a decrease in Ran GTPase-activating protein 1 (Ran Gap1) due to vasopressin. RNA interference against V1aR abolished the nuclear accumulation of MR induced by aldosterone or vasopressin. Vasopressin increased PKCα and -ß(1) expression, and aldosterone increased PKCδ and -ζ expression, but these effects were abolished with a V1aR knockdown. These results suggest that vasopressin directly regulates the nucleocytoplasmic transport of MRs via the V1aR in the intercalated cells of the collecting ducts.

The generation of rat dorsal root ganglion cell lines to identify the target of KW-7158, a novel treatment for overactive bladder.

KW-7158 is a drug candidate for the treatment of overactive bladder. Although pharmacological studies have suggested that it suppresses afferent nerve conduction, its molecular target is unknown. We herein report the establishment of dorsal root ganglion (DRG) cell lines useful for identification of the target of this compound. First, we confirmed that the target exists in rat primary DRG by [(3)H]KW-7158 binding. To establish DRG cell lines, we used DRG from transgenic rats harboring the temperature-sensitive large T-antigen. The immortalized cells were initially screened for their expression of neuronal markers, and 72 positive clones were obtained (designated as TRD cells). Next, in order to select TRD cells expressing the target of KW-7158, we measured the binding affinity and amount of the binding sites present in each clone. Most clones expressed two binding sites, one with low affinity and one with high affinity. Differential binding of KW-7158 derivatives to each site revealed that the high affinity site is pharmacologically relevant. Therefore, we successfully identified "TRD-10" which express the largest amount of the high affinity site. These cell lines will therefore be useful tools to identify the target of KW-7158.

Aldosterone requires vasopressin V1a receptors on intercalated cells to mediate acid-base homeostasis.

Both aldosterone and luminal vasopressin may contribute to the maintenance of acid-base homeostasis, but the functional relationship between these hormones is not well understood. The effects of luminal vasopressin likely result from its interaction with V1a receptors on the luminal membranes of intercalated cells in the collecting duct. Here, we found that mice lacking the V1a receptor exhibit type 4 renal tubular acidosis. The administration of the mineralocorticoid agonist fludrocortisone ameliorated the acidosis by restoring excretion of urinary ammonium via increased expression of Rhcg and H-K-ATPase and decreased expression of H-ATPase. In a cell line of intercalated cells established from transgenic rats expressing the mineralocorticoid and V1a receptors, but not V2 receptors, knockdown of the V1a receptor gene abrogated the effects of aldosterone on H-K-ATPase, Rhcg, and H-ATPase expression. These data suggest that defects in the vasopressin V1a receptor in intercalated cells can cause type 4 renal tubular acidosis and that the tubular effects of aldosterone depend on a functional V1a receptor in the intercalated cells.

Role of peroxiredoxin III in the pathogenesis of pre-eclampsia as evidenced in mice.

As a member of peroxiredoxin (Prx) family, PrxIII has been demonstrated to play an important role in scavenging intracellular reactive oxygen species (ROS). Since PrxIII knockout mice exhibited oxidative stress in placentas resembling pathophysiologic changes in placentas of human pre-eclampsia, we measured blood pressure through the carotid artery and detected oxidative status by Western blotting in pregnant mice. We did not notice hypertension in pregnant PrxIII knockout mice as compared with wild-type littermates, although endothelin-1 was over-expressed in PrxIII-deficient placentas. Our results indicate that PrxIII is not involved in pre-eclamptic development. Instead, PrxIII is an indispensable antioxidant in placentas where oxidative stress exists.

Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood-brain barrier function.

The breakdown of the blood-brain barrier (BBB) has been considered to be a key step in the disease process of a number of neurological disorders such as cerebral ischemia and Alzheimer's disease. Many in vitro BBB models derived from animal tissues have been established to elucidate the mechanism of BBB insufficiency. However, only a few human immortalized in vitro BBB models have been reported. In the present study, a temperature-sensitive SV40-T antigen was introduced to immortalize cells using a retrovirus to obtain a better human in vitro BBB model which sustains physiological properties. This endothelial cell (EC) line, termed TY08, showed a spindle-shaped morphology. The cells expressed all key tight junctional proteins, such as occludin, claudin-5, zonula occludens (ZO)-1 and ZO-2 at their cell-to-cell boundaries, and had low permeability to inulin across its monolayer. The cells also expressed various influx and efflux transporters and exhibited the functional expression of p-glycoprotein. Furthermore, the TY08 cells grew and proliferated well under the permissive temperature and stopped growing under the non-permissive temperature to serve as physiological ECs forming the BBB. Thus, conditionally immortalized TY08 cells retaining the in vivo BBB functions should facilitate analyses for determining the pathophysiology of various neurological diseases.

Establishment of a peritoneal mesothelial cell line from a transgenic rat harbouring the temperature-sensitive simian virus 40 large T-antigen gene.

BACKGROUND: Establishing a peritoneal mesothelial cell (MC) line in which the native characteristics of primary MCs are constantly maintained in vivo is of great significance for investigating their morphological and functional changes in peritoneal dialysis. We established transgenic (Tg) rats that expressed the temperature-sensitive tsA58 mutant of the simian virus 40 large T-antigen (tsSV40T), which served as a source of immortalized rat cell lines. The cells were immortalized at a permissive temperature of 33 degrees C, although they were differentiated at a non-permissive temperature of 38 degrees C. In this study, we established a novel MC line from tsSV40T Tg rats and evaluated its characteristics. METHODS: MCs were isolated from 8-week-old tsSV40T Tg rats and cloned. MCs from 8-week-old Wistar rats were used as controls. These cells were immunohistochemically and phenotypically evaluated by immunofluorescence, phase contrast and electron microscopy. The production of plasminogen activator inhibitor 1 (PAI-1) from MCs stimulated by tumour necrosis factor-alpha (TNF-alpha) was measured. RESULTS: The tsSV40T MCs showed a cobblestone-like appearance at 33 and 38 degrees C, which was similar to normal primary cultured MCs. Microvilli-like structures were observed on the cell surface by a scanning electron microscope at 33 and 38 degrees C. Wilms tumour-1 and pancytokeratin, as MC markers, were expressed at 33 and 38 degrees C. Following TNF-alpha stimulation, PAI-1 production of tsSV40T MCs was similar to that of normal primary cultured MCs. CONCLUSION: We established a novel, conditionally immortalized MC line that continuously retained the characteristics of primary cultured peritoneal MCs. This cell line might be a useful tool for various types of in vitro biological research on peritoneal dialysis.

Peroxiredoxin III-deficiency sensitizes macrophages to oxidative stress.

As a mitochondrial scavenger of reactive oxygen species (ROS), peroxiredoxin III (PrxIII) plays an important role in regulating intracellular ROS level. We previously found that PrxIII knockout (PrxIII(-/-)) mice were more sensitive than wild-type (PrxIII(+/+)) controls to intratracheal inoculation of lipopolysaccharide (LPS), but the precise mechanism remained to be obscure. In the present study, we detected the levels of ROS and tumour necrosis factor alpha (TNF-alpha) in mouse bone-marrow-derived macrophages. LPS stimulation induced transient increase of ROS production and augmentation of TNF-alpha accumulation in PrxIII(-/-) macrophages. In addition, we observed reduced viability and increased apoptosis in PrxIII(-/-) macrophages exposed to LPS. Our results provide direct evidence that PrxIII is necessary for macrophages to protect against LPS-induced oxidative stress.

p63/p51-induced onset of keratinocyte differentiation via the c-Jun N-terminal kinase pathway is counteracted by keratinocyte growth factor.

p63/p51, a homolog of the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. Because it remains unclear how p63 regulates epithelial cell differentiation, we examined the function(s) of p63 in keratinocyte differentiation through the use of a keratinocyte culture system. DeltaNp63alpha (DeltaNp51B), a p63 isoform specifically expressed in basal keratinocytes, suppressed the differentiation of specific late-stage proteins, such as filaggrin and loricrin. In contrast, DeltaNp63alpha induced keratin 1 (K1), which is expressed at the start of differentiation, via c-Jun N-terminal kinase (JNK)/AP-1 activation. However, p63 did not induce K1 expression in the basal layer in vivo, although basal keratinocytes had high levels of p63. This discrepancy was explained by the suppression of K1 expression by dermis-secreted keratinocyte growth factor. This suppression occurred via extracellular signal-related kinase (ERK) signaling, and counteracted the p63-mediated induction of K1. Thus, a precise balance between p63 and keratinocyte growth factor mediates the onset of epithelial cell differentiation, through JNK and ERK signaling. These data may provide mechanistic explanations for the pathological features of skin diseases, including psoriasis.

Establishment and functional characterization of a tracheal epithelial cell line RTEC11 from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen.

A tracheal epithelial cell line RTEC11 was established from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen. The cells grew continuously at a permissive temperature of 33 degrees C but not at a non-permissive temperature of 39 degrees C. Morphological and functional investigations demonstrated that the cells were polarized epithelial cells maintaining a regulated permeability barrier function. Interestingly, the expression levels of Muc1 (mucin 1) and Scgb1a1 (uteroglobin), non-ciliated secretory cell markers, and Tubb4 (tubulin beta 4), a ciliated cell marker, were significantly increased under the cell growth-restricted condition. Global gene expression and computational network analyses demonstrated a significant genetic network associated with cellular development and differentiation in cells cultured at the non-permissive temperature. The tracheal epithelial cell line RTEC11 with unique characteristics should be useful as an in vitro model for studies of the physiological functions and gene expression of tracheal epithelial cells.

Crucial role of peroxiredoxin III in placental antioxidant defense of mice.

We observed frequent stillbirth in peroxiredoxin III (PrxIII) knockout maternal mice. Quantitative real time PCR (qRT-PCR) and Western-blot analysis revealed increased oxidative stress in placentas that were deficient in PrxIII. We did not find significant difference between PrxIII knockout maternal mice and wild-type littermates in hematological parameters, fetal number, and embryonic development. Nevertheless, we noticed enhanced expression of PrxI in erythrocytes of pregnant knockout mice. Our results provided in vivo evidence that PrxIII played a crucial role in placental antioxidant defense. Up-regulation of PrxI might provide a compensation that protected erythrocytes against oxidative damage.

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells.

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.

Position fine-tuning of caudal primary motoneurons in the zebrafish spinal cord.

In zebrafish embryos, each myotome is typically innervated by three primary motoneurons (PMNs): the caudal primary (CaP), middle primary (MiP) and rostral primary (RoP). PMN axons first exit the spinal cord through a single exit point located at the midpoint of the overlying somite, which is formed beneath the CaP cell body and is pioneered by the CaP axon. However, the placement of CaP cell bodies with respect to corresponding somites is poorly understood. Here, we determined the early events in CaP cell positioning using neuropilin 1a (nrp1a):gfp transgenic embryos in which CaPs were specifically labeled with GFP. CaP cell bodies first exhibit an irregular pattern in presence of newly formed corresponding somites and then migrate to achieve their proper positions by axonogenesis stages. CaPs are generated in excess compared with the number of somites, and two CaPs often overlap at the same position through this process. Next, we showed that CaP cell bodies remain in the initial irregular positions after knockdown of Neuropilin1a, a component of the class III semaphorin receptor. Irregular CaP position frequently results in aberrant double exit points of motor axons, and secondary motor axons form aberrant exit points following CaP axons. Its expression pattern suggests that sema3ab regulates the CaP position. Indeed, irregular CaP positions and exit points are induced by Sema3ab knockdown, whose ectopic expression can alter the position of CaP cell bodies. Results suggest that Semaphorin-Neuropilin signaling plays an important role in position fine-tuning of CaP cell bodies to ensure proper exit points of motor axons.

Endothelial cells constituting blood-nerve barrier have highly specialized characteristics as barrier-forming cells.

In autoimmune disorders of the peripheral nervous system (PNS) such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy, breakdown of the blood-nerve barrier (BNB) has been considered as a key step in the disease process. Hence, it is important to know the cellular property of peripheral nerve microvascular endothelial cells (PnMECs) constituting the bulk of BNB. Although many in vitro models of the blood-brain barrier (BBB) have been established, very few in vitro BNB models have been reported so far. We isolated PnMECs from transgenic rats harboring the temperature-sensitive SV40 large T-antigen gene (tsA58 rat) and investigated the properties of these "barrier-forming cells". Isolated PnMECs (TR-BNBs) showed high transendothelial electrical resistance and expressed tight junction components and various types of influx as well as efflux transporters that have been reported to function at BBB. Furthermore, we confirmed the in vivo expression of various BBB-forming endothelial cell markers in the endoneurium of a rat sciatic nerve. These results suggest that PnMECs constituting the bulk of BNB have a highly specialized characteristic resembling the endothelial cells forming BBB.

Ser46 phosphorylation of p53 is not always sufficient to induce apoptosis: multiple mechanisms of regulation of p53-dependent apoptosis.

The tumor suppressor gene p53 plays a central role in determining cell fate in response to DNA damage; cells may undergo either senescence or apoptosis, depending on cell type. Phosphorylation of Serine 46 (Ser(46)) of p53 is considered to be a primary determinant for the induction of apoptosis, by selectively inducing transactivation of p53 target genes that have proapoptotic function. However, the generality of this mechanism of regulation of p53 remains a matter of debate. We investigated the role of p53 phosphorylation in adriamycin (ADR)-induced apoptosis. We found that Ser(46) was phosphorylated in four different cell lines undergoing ADR-induced senescence, as well as in two different cell lines undergoing ADR-induced apoptosis. Using alanine and glutamic acid substitution mutants of p53 Ser(46), we showed that Ser(46 )phosphorylation is not a prerequisite for induction of the proapoptotic gene AIP1. These results indicate that Ser(46) phosphorylation of p53 is not required for ADR-induced apoptosis.

Genetic networks in nonpermissive temperature-induced cell differentiation of Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen.

To identify the molecular basis by which nonpermissive temperature (NPT) induces cell differentiation in Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen, we performed global scale microarray and computational gene network analyses. In TTE3 cells, inactivation of the large T-antigen by a NPT at 39 degrees C led to cell differentiation accompanying elevation of transferrin, a marker for differentiation of Sertoli cells, and CDKN1A, a cyclin-dependent kinase inhibitor. Of the 22,690 probe sets analyzed, NPT down-regulated 498 probe sets and up-regulated 432 probe sets by >2.0-fold. Hierarchical clustering analysis showed six gene clusters. In the down-regulated cluster I, a significant genetic network including fibronectin 1 was associated with cellular growth and proliferation. In up-regulated cluster IV, a significant genetic network including CDKN1A was associated with cellular differentiation. The present results provide additional novel insights into the molecular basis of cell differentiation induced by NPT in cells.

Establishment of cell lines derived from the rat suprachiasmatic nucleus.

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.

Increased susceptibility of MER5 (peroxiredoxin III) knockout mice to LPS-induced oxidative stress.

MER5 (also called peroxiredoxin III, PrxIII) is a member of peroxiredoxin family that has antioxidant activity. The present study was performed to investigate its in vivo function using MER5 knockout mice. MER5 knockout mice were born in normal frequency and could grow to maturity, but we found that intracellular ROS levels are significantly higher in the macrophages of the knockout mice. We examined roles of MER5 function for the oxidative stress responses by intratracheal inoculation of lipopolysaccharide (LPS) to the mice. Lung inflammation such as inflammatory cell infiltration and airway wall thickening was more severely detected in the knockout mice. At the same time, oxidative damage on DNA and proteins was more strongly detected in lung tissues of the knockout mice, including 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation and protein carbonylation. The degrees of lung inflammation and oxidative damage were positively related with LPS doses. Our results indicate that MER5 knockout mice accumulated higher intracellular ROS levels, which cause LPS-induced lung injury more severely, and thus, suggested that MER5 acts as an important scavenger of reactive oxygen species (ROS) under oxidative stress.

The immortalized cell lines with differentiation potentials: their establishment and possible application.

Approximately 200 types of the cells are qualified as differentiated cells in the human body. If these different types of cells can be separated from each other (or cloned) and obtained in sufficient quantity, it will be beneficial for studying development, morphogenesis, tissue maintenance, cancer and aging, and for reconstructing functional tissues in vitro for regenerative medicine. We produced the transgenic mouse and rat harboring SV40 T-antigen gene to make the immortalized cell lines in the primary tissue culture and succeeded in establishing many functionally active cell lines from various tissues. Many immortalized cell lines from various tissues are shown to exhibit the unique characteristics of tissue functions and they should be useful as an in vitro model of various tissues for physiological and pharmacological investigations. Future application of these cells to drug screening is discussed.