Aberrant fibroblast growth factor receptor signaling in bladder and other cancers

Fibroblast growth factors (FGFs) are potent mitogens, morphogens, and inducers of angiogenesis, and FGF signaling governs the genesis of diverse tissues and organs from the earliest stages. With such fundamental embryonic and homeostatic roles, it follows that aberrant FGF signaling underlies a variety of diseases. Pathological modifications to FGF expression are known to cause salivary gland aplasia and autosomal dominant hypophosphatemic rickets, while mutations in FGF receptors (FGFRs) result in a range of skeletal dysplasias. Anomalous FGF signaling is also associated with cancer development and progression. Examples include the overexpression of FGF2 and FGF6 in prostate cancer, and FGF8 overexpression in breast and prostate cancers. Alterations in FGF signaling regulators also impact tumorigenesis, which is exemplified by the down-regulation of Sprouty 1, a negative regulator of FGF signaling, in prostate cancer. In addition, several FGFRs are mutated in human cancers (including FGFR2 in gastric cancer and FGFR3 in bladder cancer). We recently identified intriguing alterations in the FGF pathway in a novel model of bladder carcinoma that consists of a parental cell line (TSU-Pr1/T24) and two sublines with increasing metastatic potential (TSU-Pr1-B1 and TSU-Pr1-B2), which were derived successively through in vivo cycling. It was found that the increasingly metastatic sublines (TSU-Pr1-B1 and TSU-Pr1-B2) had undergone a mesenchymal to epithelial transition. FGFR2IIIc expression, which is normally expressed in mesenchymal cells, was increased in the epithelial-like TSU-Pr1-B1 and TSU-Pr1-B2 sublines and FGFR2 knock-down was associated with the reversion of cells from an epithelial to a mesenchymal phenotype. These observations suggest that modified FGF pathway signaling should be considered when studying other cancer types.     

Differential tyrosine phosphorylation of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin

The sulfated regions in heparan sulfate and heparin are known to affect fibroblast growth factor (FGF) function. We have studied the mechanism whereby heparin directs FGF-2-induced FGF receptor-1 (FGFR-1) signal transduction. FGF-2 alone stimulated maximal phosphorylation of Src homology domain 2 tyrosine phosphatase (SHP-2) and the adaptor molecule Crk, in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells expressing FGFR-1. In contrast, for phospholipase Cgamma(1) (PLCgamma(1)) and the adaptor molecule Shb to be maximally tyrosine-phosphorylated, cells had to be stimulated with both FGF-2 and heparin (100 ng/ml). Tyrosine residues 463 in the juxtamembrane domain and 766 in the C-terminal tail in FGFR-1 are known to bind Crk and PLCgamma(1), respectively. Analysis of tryptic phosphopeptide maps of FGFR-1 from cells stimulated with FGF-2 alone and FGF-2 together with heparin showed that FGF-2 alone stimulated a several-fold increase in tyrosine 463 in the juxtamembrane domain. In contrast, heparin had to be included in order for tyrosine 766 to be phosphorylated to the same fold level. Our data imply that tyrosine 463 is phosphorylated and able to transduce signals in response to FGF-2 treatment alone; furthermore, we suggest that FGFR-1 dimerization/kinase activation is stabilized by heparin.     

Effects of cis and trans regulatory variations on the expression divergence of heat shock response genes between yeast strains

Phenotypic variation among individuals in a population can be due to DNA sequence variation in protein coding regions or in regulatory elements. Recently, many studies have indicated that mutations in regulatory elements may be the major cause of phenotypic evolution. However, the mechanisms for evolutionary changes in gene expression are still not well understood. Here, we studied the relative roles of cis and trans regulatory changes in Saccharomyces cerevisiae cells to cope with heat stress. It has been found that the expression level of ~ 300 genes was induced at least two fold and that of ~ 500 genes was repressed at least two fold in response to heat shock. From the former set of genes, we randomly selected 65 genes that showed polymorphism(s) between the BY and RM strains for pyrosequencing analysis to explore the relative contributions of cis and trans regulatory variations to the expression divergence between BY and RM. Our data indicated that the expression divergence between BY and RM was mainly due to trans regulatory variations under either the normal condition or the heat stress condition. However, the relative contribution of trans regulatory variation was decreased from 76.9% to 61.5% after the heat shock stress. These results indicated that the cis regulatory variation may play an important role in the adaption to heat stress. In our data, 43.1% (28 genes) of the 65 genes showed the same trend of cis or trans variation effect after the heat shock stress, 35.4% (23 genes) showed an increased cis variation effect and 21.5% (14 genes) showed an increased trans variation effect after the heat shock stress. Thus, our data give insights into the relative roles of cis and trans variations in response to heat shock in yeast.     

Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T₃ and T₄ levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn⁺⁺ in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.     

Up-regulation of circulating hemocyte population in response to bacterial challenge is mediated by octopamine and 5-hydroxytryptamine via Rac1 signal in Spodoptera exigua

Bacterial challenge induced a significant increase in the total hemocyte population within 4 h in the beet armyworm, Spodoptera exigua. Octopamine and 5-hydroxytryptamine (5-HT) are known to play critical roles in mediating insect immune responses. This study analyzed the effects of both biogenic monoamines on mediating up-regulation of circulating hemocyte population in response to bacterial challenge. Injection of either octopamine or 5-HT induced a significant increase in the total hemocyte count in the hemolymph without any bacterial challenge. On the other hand, the monoamine antagonists, phentolamine (an octopamine antagonist) and ketanserin (a 5-HT antagonist) each suppressed the increase of the circulating hemocyte counts in response to bacterial challenge. This rapid change of circulating hemocyte population did not appear to be the result of de novo hemocyte production from the hematopoietic organ because a physical block (“ligation”) of hemolymph circulation between thorax and abdomen did not inhibit the increase of hemocyte counts in the isolated abdomen in response to bacterial challenge. The effects of the two monoamines on hemocyte numbers were not dependent on the mediatory effects of eicosanoids, because dexamethasone, an eicosanoid biosynthesis inhibitor, had no effect on the hemocyte recruitment induced by the monoamines. On the other hand, an adenylate cyclase inhibitor, NKY80, significantly impaired hemocyte mobilization in response to bacterial challenge, implying involvement of cyclic AMP in the control of hemocyte numbers. Also, a Rac1 inhibitor, NSC23766, significantly antagonized the effects of monoamines in increasing circulating hemocyte numbers. Rac1 activity was necessary to form F-actins in the hemocytes of S. exigua, where its activity showed a quantitative correlation with hemocyte-spreading behavior. This study suggests that octopamine and 5-HT mediate a rapid increase of circulating hemocyte population in response to bacterial challenge via Rac1 signal in S. exigua.     

Tibolone and its metabolites enhance tissue factor and PAI-1 expression in human endometrial stromal cells: Evidence of progestogenic effects

Tibolone is a highly effective postmenopausal hormone treatment that has its biological activity dependent on metabolism to 3alpha- and 3beta-OH tibolone, which bind solely to the estrogen receptor. Despite the high levels of estrogen receptor-binding metabolites in the circulation, the endometrium becomes atrophic, suggesting inactivation of the estrogen response in this tissue which may be due to the progestogenic activity of tibolone and the Delta-4 tibolone metabolite. We evaluated the effects of tibolone and its metabolites on tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) expression in human endometrial stromal cells (HESCs). Since TF and PAI-1 exhibit long-term in vivo and in vitro up-regulation by progestin they serve as endpoints for assessing chronic effects of progestin exposure. Confluent HESCs were primed in serum-containing medium with vehicle control, 10(-8)mol/L estradiol, 10(-7)mol/L medroxyprogesterone acetate, or 10(-8) to 10(-6)mol/L tibolone or its metabolites, then switched to a defined medium with corresponding vehicle or steroids. After 24h, ELISAs indicated that the progestin elevated TF (6.2-fold +/-3.0; p<0.05) and PAI-1 (eight-fold +/-2.1; p<0.05) levels, whereas the cells were refractory to estradiol exposure. Tibolone and Delta-4 tibolone (10(-8) to 10(-6)mol/L) were as effective as 10(-7)mol/L medroxyprogesterone acetate in enhancing TF and PAI-1 output (p<0.05). Unexpectedly, at the higher concentrations 3alpha- and 3beta-OH tibolone also elevated TF and PAI-1 expression (p<0.05). Western blotting confirmed the ELISA results. Our findings suggest that HESCs metabolize 3alpha- and 3beta-OH tibolone to tibolone and subsequently to Delta-4 tibolone, which can both stimulate the progesterone receptor. Since TF and PAI-1 promote hemostasis by complementary mechanisms, our findings account for the reduced occurrence of abnormal uterine bleeding associated with tibolone therapy.     

ABA-insensitive (ABI) 4 and ABI5 synergistically regulate DGAT1 expression in Arabidopsis seedlings under stress

Triacylglycerol (TAG) accumulation is essential for seed maturation in plants. Diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme in TAG biosynthesis. In this study, we show that TAG accumulation in Arabidopsis seedlings is correlated with environmental stress, and both ABI4 and ABI5 play important roles in regulating DGAT1 expression. Tobacco transient assays revealed the synergistic effect of ABI4 with ABI5 in regulating DGAT1 expression. Taken together, our findings indicate ABI5 is an important accessory factor with ABI4 in the activation of DGAT1 in Arabidopsis seedlings under stress.     

Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex

The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.