Rho-kinase inhibitor restores glomerular fatty acid metabolism in diabetic kidney disease.

The small GTPase Rho and its effector Rho-kinase (ROCK) are activated in the diabetic kidney, and recent studies decade have demonstrated that ROCK signaling is an integral pathway in the progression of diabetic kidney disease. We previously identified the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism in diabetic glomeruli. However, the effect of pharmacological intervention for ROCK1 is not clear. In the present study, we show that the inhibition of ROCK1 by Y-27632 and fasudil restores fatty acid oxidation in the glomeruli. Mechanistically, these compounds optimize fatty acid utilization and redox balance in mesangial cells via AMPK phosphorylation and the subsequent induction of PGC-1α. A further in vivo study showed that the inhibition of ROCK1 suppressed the downregulation of the fatty acid oxidation-related gene expression in glomeruli and mitochondrial fragmentation in the mesangial cells of db/db mice. These observations indicate that ROCK1 could be a promising therapeutic target for diabetic kidney disease through a mechanism that improves glomerular fatty acid metabolism.

Rho-associated, coiled-coil-containing protein kinase 1 regulates development of diabetic kidney disease via modulation of fatty acid metabolism.

Dysregulation of fatty acid utilization is increasingly recognized as a significant component of diabetic kidney disease. Rho-associated, coiled-coil-containing protein kinase (ROCK) is activated in the diabetic kidney, and studies over the past decade have illuminated ROCK signaling as an essential pathway in diabetic kidney disease. Here, we confirmed the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism using glomerular mesangial cells and ROCK1 knockout mice. Mesangial cells with ROCK1 deletion were protected from mitochondrial dysfunction and redox imbalance driven by transforming growth factor ß, a cytokine upregulated in diabetic glomeruli. We found that high-fat diet-induced obese ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. Mechanistically, we found that ROCK1 regulates the induction of critical mediators in fatty acid metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1α, carnitine palmitoyltransferase 1, and widespread program-associated cellular metabolism. Thus, our findings highlight ROCK1 as an important regulator of energy homeostasis in mesangial cells in the overall pathogenesis of diabetic kidney disease.

Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1.

Autophagy is the process by which intracellular components are degraded by lysosomes. It is also activated by oxidative stress; hence, autophagy is thought to be closely related to oxidative stress, one of the major causes of diabetic neuropathy. We previously reported that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) induced antioxidant enzymes and protected Schwann cells from oxidative stress. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) decreased the cell survival rate, as measured by an MTT assay in immortalized Fischer rat Schwann cells 1 (IFRS1). A DHA pretreatment significantly prevented tBHP-induced cytotoxicity. tBHP increased autophagy, which was revealed by the ratio of the initiation markers, AMP-activated protein kinase, and UNC51-like kinase phosphorylation. Conversely, the DHA pretreatment suppressed excessive tBHP-induced autophagy signaling. Autophagosomes induced by tBHP in IFRS1 cells were decreased to control levels by the DHA pretreatment whereas autolysosomes were only partially decreased. These results suggest that DHA attenuated excessive autophagy induced by oxidative stress in Schwann cells and may be useful to prevent or reduce cell death in vitro. However, its potentiality to treat diabetic neuropathy must be validated in in vivo studies.

Aldose Reductase and the Polyol Pathway in Schwann Cells: Old and New Problems.

Aldose reductase (AR) is a member of the reduced nicotinamide adenosine dinucleotide phosphate (NADPH)-dependent aldo-keto reductase superfamily. It is also the rate-limiting enzyme of the polyol pathway, catalyzing the conversion of glucose to sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase. AR is highly expressed by Schwann cells in the peripheral nervous system (PNS). The excess glucose flux through AR of the polyol pathway under hyperglycemic conditions has been suggested to play a critical role in the development and progression of diabetic peripheral neuropathy (DPN). Despite the intensive basic and clinical studies over the past four decades, the significance of AR over-activation as the pathogenic mechanism of DPN remains to be elucidated. Moreover, the expected efficacy of some AR inhibitors in patients with DPN has been unsatisfactory, which prompted us to further investigate and review the understanding of the physiological and pathological roles of AR in the PNS. Particularly, the investigation of AR and the polyol pathway using immortalized Schwann cells established from normal and AR-deficient mice could shed light on the causal relationship between the metabolic abnormalities of Schwann cells and discordance of axon-Schwann cell interplay in DPN, and led to the development of better therapeutic strategies against DPN.

Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro.

Besides its insulinotropic actions on pancreatic ß cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron-IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.

Glycolaldehyde induces sensory neuron death through activation of the c-Jun N-terminal kinase and p-38 MAP kinase pathways.

Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10 < 100 < 250 < 500 µM; 6 h < 24 h). Treatment with 500 µM GA significantly up-regulated the phosphorylation of c-jun N-terminal kinase (JNK) and p-38 mitogen-activated kinase (p-38 MAPK) in ND7/23 cells. Furthermore, GA-induced ND7/23 cell death was significantly inhibited due to co-treatment with 10 µM of the JNK inhibitor SP600125 or the p-38 MAPK inhibitor SB239063. These findings suggest the involvement of JNK and p-38 MAPK-signaling pathways in GA-induced neuronal cell death and that enhanced GA production under diabetic conditions might be involved in the pathogenesis of diabetic neuropathy.

Drug-Induced Demyelinating Neuropathies.

A large variety of drugs have been reported to cause peripheral neuropathies as dose-limiting adverse effects; however, most of them primarily affect axons and/or neuronal cell bodies rather than Schwann cells and/or myelin sheaths. In this chapter, we focus on the drugs that seem to elicit the neuropathies with schwannopathy and/or myelinopathy-predominant phenotypes, such as amiodarone, dichloroacetate, and tumor necrosis factor-α antagonists. Although the pathogenesis of demyelination induced by these drugs remain largely obscure, the recent in vivo and in vitro studies have implicated the involvement of metabolic abnormalities and impaired autophagy in Schwann cells and immune system disorders in the disruption of neuron-Schwann cell contact and interactions.

A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR.

Establishment of a myelinating co-culture system with a motor neuron-like cell line NSC-34 and an adult rat Schwann cell line IFRS1.

Co-culture models of neurons and Schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained by primary culture with embryonic or neonatal animals. A spontaneously immortalized Schwann cell line IFRS1 from long-term cultures of adult Fischer rat peripheral nerves has been shown to retain fundamental ability to myelinate neurites in co-cultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Our current investigation focuses on the establishment of stable co-culture system with IFRS1 cells and NSC-34 motor neuron-like cells. NSC-34 cells were seeded at a low density (2 × 103/cm2) and maintained for 5-7 days in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (BDNF; 10 ng/mL). Upon observation of neurite outgrowth under a phase-contrast microscope, the NSC-34 cells were exposed to an anti-mitotic agent mitomycin C (1 µg/mL) for 12-16 h, then co-cultured with IFRS1 cells (2 × 104/cm2), and maintained in serum-containing medium supplemented with ascorbic acid (50 µg/mL), BDNF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Double immunofluorescence staining carried out at day 28 of the co-culture showed myelin protein (P0 or PMP22)-immunoreactive IFRS1 cells surrounding the ßIII tubulin-immunoreactive neurites. This co-culture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g., amyotrophic lateral sclerosis, Charcot-Marie-Tooth diseases, and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them.

TGFß signaling reinforces pericyte properties of the non-endocrine mouse pituitary cell line TtT/GF.

The non-endocrine TtT/GF mouse pituitary cell line was derived from radiothyroidectomy-induced pituitary adenoma. In addition to morphological characteristics, because the cells are S100ß-positive, they have been accepted as a model of folliculostellate cells. However, our recent microarray analysis indicated that, in contrast to folliculostellate cells, TtT/GF cells might not be terminally differentiated, as they share some properties with stem/progenitor cells, vascular endothelial cells and pericytes. The present study investigates whether transforming growth factor beta (TGFß) can elicit further differentiation of these cells. The results showed that canonical (Tgfbr1 and Tgfbr2) and non-canonical TGFß receptors (Tgfbr3) as well as all TGFß ligands (Tgfb1-3) were present in TtT/GF cells, based on reverse transcription PCR. SMAD2, an intercellular signaling molecule of the TGFß pathway, was localized in the nucleus upon TGFß signaling. Furthermore, TGFß induced cell colony formation, which was completely blocked by a TGFß receptor I inhibitor (SB431542). Real-time PCR analysis indicated that TGFß downregulated stem cell markers (Sox2 and Cd34) and upregulated pericyte markers (Nestin and Ng2). Double immunohistochemistry using mouse pituitary tissue confirmed the presence of NESTIN/NG2 double-positive cells in perivascular areas where pericytes are localized. Our results suggest that TtT/GF cells are responsive to TGFß signaling, which is associated with cell colony formation and pericyte differentiation. As pericytes have been shown to regulate angiogenesis, tumorigenesis and stem/progenitor cells in other tissues, TtT/GF cells could be a useful model to study the role of pituitary pericytes in physiological and pathological processes.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone-induced schwannopathy.

Amiodarone hydrochloride (AMD), an anti-arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor-primed PC12 cells. Treatment with AMD (1, 5, and 10 µm) induced time- and dose-dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2-related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3-II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD-induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time- and dose-dependent manner. These findings suggest that AMD-induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

Expression studies of neuronatin in prenatal and postnatal rat pituitary.

The pituitary gland, an indispensable endocrine organ that synthesizes and secretes pituitary hormones, develops with the support of many factors. Among them, neuronatin (NNAT), which was discovered in the neonatal mouse brain as a factor involved in neural development, has subsequently been revealed to be coded by an abundantly expressing gene in the pituitary gland but its role remains elusive. We analyze the expression profile of Nnat and the localization of its product during rat pituitary development. The level of Nnat expression was high during the embryonic period but remarkably decreased after birth. Immunohistochemistry demonstrated that NNAT appeared in the SOX2-positive stem/progenitor cells in the developing pituitary primordium on rat embryonic day 11.5 (E11.5) and later in the majority of SOX2/PROP1 double-positive cells on E13.5. Thereafter, during pituitary embryonic development, Nnat expression was observed in some stem/progenitor cells, proliferating cells and terminally differentiating cells. In postnatal pituitaries, NNAT-positive cells decreased in number, with most coexpressing Sox2 or Pit1, suggesting a similar role for NNAT to that during the embryonic period. NNAT was widely localized in mitochondria, peroxisomes and lysosomes, in addition to the endoplasmic reticulum but not in the Golgi. The present study thus demonstrated the variability in expression of NNAT-positive cells in rat embryonic and postnatal pituitaries and the intracellular localization of NNAT. Further investigations to obtain functional evidence for NNAT are a prerequisite.

Expression of Slug in S100ß-protein-positive cells of postnatal developing rat anterior pituitary gland.

Among heterogeneous S100ß-protein-positive (S100ß-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100ß-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100ß-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100ß-promoter has allowed us to observe living S100ß-positive cells. In the present study, we first confirmed that living S100ß-positive cells in tissue cultures of S100ß-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100ß-positive cells. Interestingly, we detected Slug expression in S100ß-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100ß-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100ß-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

GFP-expressing S100ß-positive cells of the rat anterior pituitary differentiate into hormone-producing cells.

Some non-endocrine cells in the pituitary anterior lobe are responsible for providing stem/progenitor cells to maintain hormone-producing cells. In particular, cells expressing S100ß protein, a calcium-binding protein, have been hypothesized to be a pituitary cell resource. Accumulating data have revealed that S100ß-positive cells comprise heterogeneous populations and some of them certainly show stem/progenitor characteristics in vivo. Hence, we examine whether S100ß-positive cells have the capacity to differentiate into endocrine cells, by means of in vivo and in vitro experiments on transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of the S100ß promoter. Immunohistochemistry of the pituitary confirmed that some S100ß-positive cells expressed SOX2 (SRY [sex-determining region Y]-box 2) and had proliferative activity. Dispersed anterior lobe cells were observed by time-lapse microscopy, followed by immunostaining for hormone and pituitary-transcription-factor1 (PIT1). First, the dispersed anterior lobe cells were immunostained by an antibody against SOX2. S100ß-protein co-localizes with SOX2 (about 89 %). Although 44 of 134 S100ß-positive cells traced were proliferative but negative to any hormones, 14 cells were positive for one of the pituitary hormones and/or PIT1, confirming the presence of all types of hormone-producing cells. Notably, GFP-fluorescence appeared in two hormone-positive cells during culture. On the other hand, we observed hormone-producing cells that were not positive for S100ß at the end of the time-lapse study, despite being initially positive. These findings suggest that S100ß-positive cells cultured from the anterior lobe are capable of developing into hormone-producing cells, although this happens relatively infrequently.

Characterization of murine pituitary-derived cell lines Tpit/F1, Tpit/E and TtT/GF.

The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100ß and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.

[Pathogenic Mechanisms of Diabetic Neuropathy].

Diabetes stands as the predominant cause of peripheral neuropathy, and diabetic neuropathy (DN) is an early-onset and most frequent complication of diabetes. Distal symmetric polyneuropathy is the major form of DN; however, various patterns of nerve injury can manifest. Growing evidence suggests that hyperglycemia-related metabolic disorders in neurons, Schwann cells, and vascular endothelial cells play a major role in the development and progression of DN; however, its pathogenesis and development of disease-modifying therapies warrant further investigation. Herein, recent studies regarding the possible pathogenic factors of DN (polyol and other collateral glycolysis pathways, glycation, oxidative stress, Rho/Rho kinase signaling pathways, etc.) and therapeutic strategies targeting these factors are introduced.

Temporospatial gene expression of Prx1 and Prx2 is involved in morphogenesis of cranial placode-derived tissues through epithelio-mesenchymal interaction during rat embryogenesis.

Paired-related homeobox transcription factors, PRX1 and PRX2, are verified to play essential roles in limb, heart and craniofacial development by analyses of knockout animals. Their gene expression in the embryonic primordia derived from the mesoderm and neural crest is confirmed by in situ hybridization. Nevertheless, a detailed localization of PRX1 and PRX2 was not carried out because of a lack of specific antibodies for each factor. We have previously confirmed the presence of PRX proteins in rat embryonic pituitary by using an antibody that recognizes both PRX1 and PRX2. However, the pituitary originates in the cranial placodes, not the mesoderm or neural crest. In this study, we analyze the temporospatial distribution of PRX1 and PRX2 with novel antibodies specific for each factor, together with a stem/progenitor marker SOX2 (sex-determining region Y-box 2) in the primordia formed by epithelio-mesenchymal interaction. We observe immunoreactive signals of both PRX proteins in rat embryo, showing a similar pattern to that obtained by in situ hybridization. In early embryogenesis, PRX proteins are not co-localized with SOX2 but PRX2 and/or PRX1-positive cells are present in the border or periphery of SOX2-positive primordia originating in the cranial placode. During advanced embryogenesis, either PRX2-positive cells become condensed in the border of SOX2-positive cells or PRX1 and/or PRX2 become co-localized with SOX2. Our results suggest that PRX proteins, especially PRX2, play a role in the morphogenesis of the primordial tissues formed by the epithelio-mesenchymal interaction and that neural crest cells contribute to the morphogenesis of tissues derived from the cranial placode.

Three-dimensional studies of Prop1-expressing cells in the rat pituitary just before birth.

Recently, we demonstrated that differentiation was underway as early as embryonic day (E) 13.5 in the lateral region of the rat pituitary primordium. In this study, we analyze the heterogeneous property of cells in the pituitary at E21.5 (just before birth) leading to its biological function with the differentiation and expansion of tissue. The three-dimensional structure of the pituitary at E21.5 was built up from measurements taken from many DAPI-stained sections and cell populations positive to the stem/progenitor marker SOX2, pituitary-specific transcription factor PROP1 and paired-related homeodomain transcription factor PRX. At E21.5, the pituitary, composed of anterior and intermediate lobes, showed a flattened chestnut shape with dimensions of about 500 µm (dorsoventral axis) by 2500 µm (left-right axis) by 850 µm (rostrocaudal axis) and consisted in approximately 113,500, 16,000 and 14,800 cells in the anterior, intermediate and posterior lobes, respectively. Five cell types were observed expressing Sox2, Prop1 and Prx; these were heterogeneously distributed in the mediolateral and dorsoventral axes. In the anterior lobe, the marginal cell layer (MCL) was mostly occupied by stem/progenitor cells positive for SOX2, with the co-expression of Prop1 and/or Prx, whereas more SOX2-single-positive cells than those for PROP1 and PRX were scattered in the parenchyma. PRX-positive cells of mesenchymal origin invaded the parenchyma, together with PECAM- and NESTIN-positive cells, indicating the advance of vasculogenesis. Thus, marked developmental progress occurs regarding the transition of stem/progenitor cells in the MCL and regarding vasculogenesis in the parenchyma during the prenatal pituitary growth wave.

Coxsackievirus and adenovirus receptor-positive cells compose the putative stem/progenitor cell niches in the marginal cell layer and parenchyma of the rat anterior pituitary.

The pituitary gland is a slow generative tissue but actively responds to demands by changing homeostasis. The marginal cell layer (MCL) facing the residual lumen has long been indicated as a stem/progenitor cell niche of the pituitary. On the other hand, the coxsackievirus and adenovirus receptor (CAR), which localizes at the tight-junction of the polarized epithelium, is known to participate in the development, differentiation and regeneration of specified tissues. The present study attempts to characterize the cells lining the MCL during pituitary development by immunohistochemistry of CAR. Consequently, we found that CAR localizes in an apical surface of the single cell layer facing the oral cavity in the invaginating oral epithelium on rat embryonic day (E) 11.5. On E13.5, when this single layer constructs the MCL in the pituitary primordium Rathke's pouch, CAR-positive cells occupied the MCL and this localization pattern of CAR was persistently maintained throughout life. Moreover, clusters of CAR-positive cells were also found in the parenchyma. CAR-positive cells were positive for stem/progenitor cell markers sex-determining region Y-box 2 (SOX2) and epithelial calcium-dependent adhesion (E-cadherin). However, prior to the postnatal growth wave, cells positive for CAR in the basolateral surface constructed multiple cell layers beneath the MCL and cell-type transition to a putative migratory cell phenotype by fading of SOX2 and E-cadherin occurred, suggesting the composition of new putative niches in the parenchyma. These data, together with our previous reports, suggest that CAR-positive cells are pituitary stem/progenitor cells and compose putative stem/progenitor cell niches in the MCL and parenchyma.

Characterization of a pituitary-tumor-derived cell line, TtT/GF, that expresses Hoechst efflux ABC transporter subfamily G2 and stem cell antigen 1.

The anterior lobe of the pituitary gland is composed of five types of endocrine cells and of non-endocrine folliculo-stellate cells that produce various local signaling molecules. The TtT/GF cell line is derived from pituitary tumors, produces no hormones and has folliculo-stellate cell-like characteristics. The biological function of TtT/GF cells remains elusive but several properties have been postulated (support of endocrine cells, control of cell proliferation, scavenger function). Recently, we observed that TtT/GF cells have high resistance to the antibiotic G418 and low influx for Hoechst 33342, indicating the presence of ATP-binding cassette (ABC) transporters that efflux multiple drugs, i.e., a property similar to that of stem/progenitor cells. Therefore, we examine TtT/GF cells for the presence of ABC transporters, for the efflux ability of Hoechst 33342 and for those genes characteristic of TtT/GF cells. Real-time polymerase chain reaction (PCR) for ABC transporters demonstrated that Abcb1a, Abcb1b and Abcg2, regarded as stem cell markers, were characteristically expressed in TtT/GF cells but not in Tpit/F1 and LßT2 cells. Furthermore, the remarkable low-efflux ability of Hoechst 33342 from TtT/GF cells was confirmed by using inhibitors and contrasted with the abilities of Tpit/F1 and LßT2 cells. The high and specific expression of stem cell antigen 1 (Sca1) in TtT/GF cells was confirmed by real-time PCR. We also demonstrated those genes that are expressed abundantly and characteristically in TtT/GF, suggesting that TtT/GF cells have unique characteristics similar to those of stem/progenitor cells of endothelial or mesenchymal origin. Thus, the present study has revealed an intriguing property of TtT/GF cells, providing a new clue for an understanding of the function of this cell line.