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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposed to pregnant or lactational mother impairs the reproduction and development of the pups. The defect is a serious problem, because it is caused by TCDD at much lower doses than that needed for acute toxicity in the mother. However, the toxic mechanism underlying the defect remains to be obscure. We have previously revealed that maternal exposure to TCDD (1 microg/kg) causes a reduction in luteinizing hormone in the fetal pituitary, leading to the reduced expression of testicular steroidogenic proteins such as steroidogenic acute-regulatory protein (StAR) and cytochrome P450 (CYP) 17. In addition, we have provided evidence that such a reduction imprints defects in sexual behaviors at adulthood. In this study, we investigated TCDD effect on fetal steroidogenesis in the extra-gonadal tissues. Even when pregnant Wistar rats at gestational day (GD) 15 were orally treated with TCDD (0.25, 1 or 3 microg/kg), neither expression of StAR nor CYP17 mRNA was affected in the adrenal gland, placenta and hypothalamus of male fetuses (GD20). However, TCDD induced placental StAR (3 microg/kg) and adrenal CYP17 mRNAs (0.25 microg/kg) in female fetuses. Therefore, our study suggests that while TCDD gives damage to male fetal steroidogenesis in a testis-specific manner, the dioxin enhances the steroidogenesis of the fetal adrenal gland and placenta in females. Thus, the mechanism whereby TCDD exerts its endocrine-disrupting properties is considered to differ, at least partially, between male and female fetuses.

A number of studies have reported that reproductive and developmental disorders are produced by prenatal or postnatal exposure to dioxins such as 2,3,7,8-tertachlorodibenzo-p-dioxin (TCDD). These effects would be more serious compared with other toxicities with dioxins, because those injuries appear at much lesser doses than those needed for acute toxicity. Although the mechanisms regulating reproductive and developmental disorders are still unclear, we have recently reported that maternal exposure to TCDD causes the suppression of fetal testicular steroidogenic enzymes in rats. In the present study, we examined whether the same occurs in mice, using C57BL/6J and DBA/2N strains. The result showed that TCDD does not show any effect on the expression of steroidogenic acute regulatory protein mRNA in both strains. To the best of our knowledge, abnormal sexual behavior by fetal exposure to TCDD has never been reported in mice. Therefore, our result supports a possibility that abnormal sex behavior by exposure to TCDD at the fetal stages occurs in rats but not in mice. In addition, the data obtained suggest that the suppression of fetal testicular steroidogenic enzymes is, at least, one of the key mechanisms for impaired sex behavior induced by dioxin.

Recent studies have demonstrated that klotho protein plays a role in calcium/phosphate homeostasis. Urakawa et al demonstrated that the concerted action of klotho and fibroblast growth factor receptor 1 reconstitutes the fibroblast growth factor 23 (FGF23) receptor (Nature 444,770,2006). We investigated the correlation between hyperphosphatemia and Pi transport activity in klotho mutant mice (kl/kl mice). kl/kl mice displayed hyperphosphatemia, high plasma 1,25 (OH) (2)D(3) levels, increased activity of the renal and intestinal sodium-dependent P (i) cotransporters, and increased levels of the type II a, type II b, and type II c transporter proteins compared with wild-type mice. Interestingly, transcript levels of the type II a/type II c transporter mRNA abundance, but not transcripts levels of type II b transporter mRNA, were markedly decreased in kl/kl mice compared with wild-type mice. Furthermore, plasma FGF23 levels were 150-fold higher in kl/kl mice than in wild-type mice. Feeding of a low-P (i) diet induced the expression of klotho protein and decreased plasma FGF23 levels in kl/kl mice, whereas colchicine treatment experiments revealed evidence of abnormal membrane trafficking of the type IIa transporter in kl/kl mice. These results indicate that hyperphosphatemia in klotho mice is due to dysregulation of expression and trafficking of the renal type II a/ II c transporters rather than to intestinal P (i) uptake.

SREBP family have been recently established as bHLH type transcription factors governing lipid synthesis. While SREBP-2 regulates expression of genes involved in cholesterol biosynthesis and LDL receptor, SREBP-1c controls fatty acid synthesis. Cellular cholesterol is regulated by feedback system where SCAP/Insig system regulates cleavage of SREBP-2 for its activation depending upon cellular cholesterol demand. Meanwhile, SREBP-1c is nutritionally regulated, excess energy intake could activate hepatic SREBP-1c expression leading to formation of remnant lipoproteins and hepatic insulin resistance through suppression of IRS-2 expression. Hepatic SREBP-1c could be deeply involved in metabolic syndrome. Thus, SREBP-1c could be a therapeutic target to treat metabolic syndrome. Polyunsaturated fatty acids suppress SREBP-1c and suppress lipogenesis.

Tooth is a composite of the three different biological mineralized tissues (dentin, enamel, and cementum) that acquired supreme mechanical properties and function necessary for the mechanical digestion of the food throughout life. The underlining mechanisms of mineralization of each of these three components are unique and different from each other. Here the focus was placed on the mineralization process of dentin and enamel of the teeth of vertebrates.

The visceral glomerular epithelial cells or podocytes of the renal glomerulus are uniquely characterized by a large cell body and numerous foot processes. These foot processes are kept wide open to facilitate passage of glomerular filtrate and are held together tenuous slit diaphragms that bridge the filtration slits. By contrast, nephrosis results in the apical displacement of the glomerular filtration slit membrane by newly formed intercellular occluding-type junctions. It is still unknown what kind of proteins are involved in the morphological changes of podocytes in nephrotic conditions. Our data show that one isoform of ZO-1 found exclusively in the tight junction(TJ) is expressed in the highly specialized slit diaphragm. In nephrotic rats induced by puromycin aminonucleoside(PAN) treatment or perfusion with polycations such as protamine sulfate(PS), the slit diaphragms are displaced by occluding-type junctions. In these cases, ZO-1 was concentrated along both the newly formed occluding-type junctions and the remaining slit diaphragms. Also, perfusion of glomeruli with polycations induces tyrosine phosphorylation of some proteins including ZO-1 within 15 min. What kind of molecules contribute to such a signal transduction? A possible candidate is podocalyxin, which is the major sialoglycoprotein of the rat glomerular epithelial cell. We cloned rat podocalyxin gene and analyzed its molecular nature. Furthermore, we have made a number of monoclonal antibodies against rat podocytes. Monoclonal antibody P-31 we made recognizes a novel intermediate filament-associated protein(p250) in rat podocytes. Our data suggest that p250 would play a important role in the organization of the intermediate filament network in both normal and diseased conditions.

Type 2 diabetes is characterized by insulin resistance in skeletal muscle. Since the molecular mechanism of insulin resistance is still unknown, insulin receptor dysfunction including abnormal IRS-1 phosphorylation is considered to be responsible for insulin resistance in some pathological states. Obesity is one of major factors to induce insulin receptor dysfunction. Regarding the mechanism of insulin resistance related obesity, the increased expression of Tumor necrosis factor alpha and abnormality in PTPase in skeletal muscle are postulated. As well as obesity, prolonged hyperglycemia, dyslipidemia and hypertension also induce the impairment of insulin receptor function. Therefore, the enhancement of insulin sensitivity by modulating these factors is a possible treatment modality in insulin resistant states.

Purpose: To understand the interaction of corneal epithelial cells with laminin, fibronectin, and collagen type IV, major components of basement membrane, we investigated whether tyrosine phosphorylation of paxillin was increased during attachment of these cells to matrix proteins. Paxillin is one of the focal adhesion proteins and it is tyrosine phosphorylated during cell adhesion., Methods: SV 40-transformed human corneal epithelial (HCE) cells were plated on these extracellular matrix proteins and incubated. The cellular lysates were submitted to immunoprecipitation and western blotting to determine tyrosine phosphorylation of paxillin., Results: When the cells were plated on laminin matrix, the stained band indicating tyrosine phosphorylated paxillin increased in proportion to cultivation periods. The increase was significant at 6 to 24 hours of cultivation. When HCE cells were cultured on various concentrations of laminin, paxillin was up-regulated in phosphorylation in a dose-dependent fashion. On a fibronectin matrix, tyrosine phosphorylation of paxillin increased in a time-dependent fashion and peak time point was 6 hours of cultivation. Paxillin was up-regulated in tyrosine phosphorylation in direct relation with the fibronectin concentration. On a type IV collagen matrix, tyrosine phosphorylation of paxillin increased in relation to time, but not so rapidly as cultures on a laminin or fibronectin matrix., Conclusion: Differential tyrosine phosphorylation of paxillin may have been caused by different extracellular matrix proteins.

Interferon Regulatory Factor-1 (IRF-1) acts as a transcriptional activator in interferon system and as a tumor suppressor. The loss of functional IRF-1 has been shown in approximately 30% of patients with myelodysplastic syndrome (MDS) and overt leukemia from MDS. Here we report an alternative mechanism of inactivation of IRF-1 activity. We identified an IRF-1 binding protein (IRF-BP). Protein sequencing revealed that IRF-BP was identical to nucleophosmin (NPM), a nucleolar phosphoprotein. Functional analysis showed that IRF-BP inhibited DNA-binding and transcriptional activity of IRF-1. Moreover when we examined several leukemia samples, the level of IRF-BP mRNA was increased. These results are consistent with the hypothesis that IRF-BP binds to IRF-1, and that overexpression of IRF-BP in leukemias leads to escape from IRF-1-regulated growth control. This hypothesis was also supported by the fact that overexpression of IRF-BP in NIH 3T3 cells rendered cells transformed.

Focal adhesion kinase (p125FAK) is a novel non-receptor cytosolic tyrosine kinase which is activated through the phosphorylation of its tyrosine residue by ligands that bind to integrins and ligands that activate protein kinase C (PKC). In diabetic glomeruli, extracellular matrix proteins such as fibronectin, laminin and type IV collagen, which bind to integrins, were found to be increased in the mesangial area. Furthermore, PKC was shown to be activated in diabetic glomeruli. These changes might be able to cause the activation of p125FAK in diabetic glomeruli. To test this hypothesis, we examined tyrosine phosphorylation of p125FAK and paxillin, a proposed substrate of p125FAK, in glomeruli isolated from streptozotocin (STZ)-induced diabetic rats. Tyrosine phosphorylation of p125FAK or paxillin was evaluated by immunoblot analysis using anti-phosphotyrosine antibody after immunoprecipitation with anti-p125FAK or anti-paxillin antibody. Three and seven weeks after STZ injection, tyrosine phosphorylation of both p125FAK and paxillin was increased in diabetic glomeruli. The increase in tyrosine phosphorylation of p125FAK and paxillin was not observed in glomeruli from diabetic rats treated with insulin. To investigate the mechanism of increase in tyrosine phosphorylation of p125FAK, we examined tyrosine phosphorylation of p125FAK in mesangial cells plated on a fibronectin-coated dish or cultured under conditions of high glucose concentration (conditions under which PKC can be activated). Attachment of the cells to fibronectin induced tyrosine phosphorylation of p125FAK, while a high glucose concentration did not modulate tyrosine phosphorylation of p125FAK. In conclusion, tyrosine phosphorylation of p125FAK and paxillin was increased in diabetic glomeruli and these alternations may have been caused by changes in extracellular matrix proteins in diabetes.

IRS-1 (insulin receptor substrate-1) is a major substrate for the insulin receptor tyrosine kinase. After phosphorylation by the insulin receptor, IRS-1 binds to the specific molecules which possess SH2 (src homology 2) domain such as 85 kDa subunit of phosphatidylinositol 3 kinase and may mediate insulin signals. The regulation of IRS-1 has been analyzed in animal models of insulin resistance, and its mechanism has been studied in culture cells. In animal models of insulin resistance, phosphorylation of IRS-1 was mainly regulated by the insulin receptor tyrosine kinase both in liver and muscle. However, IRS-1 protein level was differently regulated in muscle and liver. In muscle, IRS-1 protein decreased with dexamethasone treatment and in hypoinsulinemic states such as starvation and streptozotosine-induced diabetes and showed no change in hyperinsulinemic states such as obesity. In liver, IRS-1 protein increased with dexamethasone treatment and hypoinsulinemic states and decreased in hyperinsulinemic states. In cultured cell such as 3T3-L1 or 3T3-F442A adipocytes, IRS-1 was negatively regulated both by insulin and dexamethasone by different mechanisms. Insulin regulates the IRS-1 expression at protein level mainly by decreasing the half life of IRS-1 protein, and dexamethasone regulates it at mRNA level mainly by decreasing the half life of IRS-1 mRNA.

Insulin induces a wide variety of growth and metabolic response in many cell types. Insulin initiates its biological effects by activation of tyrosine kinase in the beta-subunit and phosphorylates several proteins, such as insulin receptor substrate-1 (IRS-1), Shc. thereby activating phosphatidyl inositol 3-kinase activity, and ras activity. MAP kinase cascade activated by ras, 70kDaS6 kinase lying downstream of PI3-kinase, and the regulation of glycogen synthase have been discussed.

IRS-1 (insulin receptor substrate-1) is a major substrate of the insulin receptor. Rat and human IRS-1 cDNAs, and human and mouse IRS-1 genes have been cloned so far. They show high homology in nucleic acids and amino acids levels, which indicate the high conservation of IRS-1 across the species. Interestingly, the entire coding region is contained in the 1st exon in the IRS-1 gene. The promoter of the mouse IRS-1 gene lacks TATA and CAAT boxes but contains 9 potential Spl binding sites, indicating that IRS-1 is a "housekeeping" gene. By deletion analysis, two positively and two negatively regulating fragments are identified in the promoter. In cultured adipocytes, insulin and dexamethasone down regulate IRS-1 expression by different mechanisms. Insulin down regulates at the post-translational level by shortening the protein half life, and dexamethasone down regulates at the post-transcriptional level mainly by shortening the mRNA half life.

Several lines of evidence have suggested that protein phosphorylation and dephosphorylation may play an important role in the regulation of metabolism and signal transduction processes. In our present study, bovine and frog retinas were incubated in Krebs' solution containing [32P] H3PO4 for the labelling of all phosphoproteins. Then, photoreceptor outer segments were isolated from each retina, and further incubated under dark or light conditions. In such conditions, several phosphoproteins were dark- or light-dependently dephosphorylated. Interestingly, the light-dependent dephosphorylated 39 kDa protein as well as the 35-36 kDa protein was commonly observed in both bovine and frog retinas. The 35-36 kDa protein is considered to be the same as the 33 kDa protein that has previously been shown to be phosphorylated light-dependently, whereas 39 kDa protein is thought to be a novel protein that undergoes light-dependent dephosphorylation in retinal photoreceptor outer segments. Thus, these proteins were thought to have significant roles in the visual transduction processes.

Phosphoproteins were partially purified from vitamin D-deficient and various aged chicken bones. The bones were first extracted with EDTA and fractionated using DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 molecular sieving chromatography. The major phosphoproteins were eluted in two different peaks on a DEAE chromatogram. The front peak contained low phosphorylated proteins and the other contained highly phosphorylated proteins. The total amount of phosphoproteins in vitamin D-deficient bone was lower than that in normal bone. The decrease of low phosphorylated proteins was especially obvious. On the other hand, molecular weights, amino acid compositions and the degree of phosphorylation were almost the same in both normal and vitamin D-deficient chick bone. The total extracted weight of phosphoproteins and the molecular weights on SDS-PAGE of partially purified phosphoproteins in each aged chick bone decreased with aging. These results suggest that the production of phosphoproteins is regulated by vitamin D and the degradation of phosphoproteins is an important factor in the progress and the maturation of calcification.

Development, growth, maturation and aging processes of secretory cells of rat salivary glands progress mainly after birth. Nuclear non-histone proteins, phosphorylated actively and reversively, have an important role as regulatory molecules of gene activity and have a possibility to bring about specific changes in these cellular processes. We examined in the present study the age-dependent changes in the phosphorylation of non-histone proteins of rat salivary glands. Nuclei purified from submandibular and parotid glands of 8-week-old rats rapidly incorporated 32P from gamma-32P-ATP into the nuclear phosphoproteins and reached equilibrium within 9 min. A preponderant amount of the 32P was present in non-histone proteins. The levels of phosphorylation of non-histone proteins in salivary gland nuclei increased rapidly after birth, reaching a maximum in both gland nuclei of 4-week-old rats and then decreasing to the levels observed in submandibular and parotid gland nuclei from 20 and 16-week-old rats, respectively. These levels were still maintained in nuclei from aged rats. Moreover, age-dependent changes in the protein kinase activity of submandibular and parotid gland nuclei were linked up with the changes in the phosphorylation of non-histone proteins. However, changes were not observed in the phosphorylation of histone proteins after birth. These results suggest that protein kinase activity in salivary gland nuclei may have an important role on age-dependent changes in cell function, mediated through the control of the phosphorylation of non-histone proteins.