Topical Application of BMS-509744, a Selective Inhibitor of Interleukin-2-Inducible T Cell Kinase, Ameliorates Imiquimod-Induced Skin Inflammation in Mice.

Psoriasis is an immune disorder-related inflammatory skin disease. Recent studies have suggested a contribution of T cell activation in the pathogenesis of psoriasis. Interleukin-2 (IL-2)-inducible T cell kinase (ITK) regulates T cell activation, including proliferation, and cytokine production. In this study, we investigated the effect of the topically administered selective ITK inhibitor BMS-509744 on imiquimod (IMQ)-induced psoriasis-like skin inflammation in mice. Topically administered BMS-509744 ameliorated IMQ-induced psoriasis-like skin inflammation as shown by decreased skin lesions, epidermal thickening, and cell infiltration into the dermis. These suppressive effects occurred with lower numbers of cluster of differentiation antigen-3+ (CD3+) T cells and T helper subset 17 (Th17)-related cytokine expression in IMQ-treated skin. IMQ-induced upregulation of proinflammatory cytokine expression was also inhibited by topical application of BMS-509744 in IMQ-treated skin. Our report showed for the first time that topical application of BMS-509744 ameliorated psoriasis-like skin inflammation in mice, which is likely mediated by the inhibition of T cell activation in the skin lesions.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Regulation of the expression and activity of glucose and lactic acid metabolism-related genes by protein kinase C in skeletal muscle cells.

Protein kinase C (PKC) modulators are very attractive therapeutic targets in cancer. Since most cancer cells display increased glycolysis, elucidations of the effects of PKC activation on glycolysis is necessary for the development of effective medicine. In the present study, to clarify the role of PKC in the regulation of glycolysis, we examined the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, on the expression and activity of glucose and lactic acid metabolism-related genes in human rhabdomyosarcoma cells (RD cells). In parallel to increases in glucose uptake and mRNA levels of glucose transporters (GLUTs) induced by PMA treatment for 6 h, the hexokinase (HK) mRNA level and activity were also significantly increased in RD cells. On the other hand, a significant increase in lactate dehydrogenase (LDH) mRNA level and activity was seen when the cells were incubated with PMA for 24 h, but not for 6 or 12 h, and was associated with lactic acid production. These effects by PMA treatment were markedly suppressed by Bisindolylmaleimide (BIM), a PKC inhibitor. Furthermore, chetomin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, completely abrogated the increment of LDH mRNA level and activity as well as monocarboxylate transporter (MCT) 4, a lactic acid efflux transporter. In conclusion, we found that HK and LDH activity induced by PKC activation was associated with the glucose uptake and lactic acid level and that LDH and MCT4 are modulated by a common factor, HIF-1.

Induction of cyclooxygenase-2 expression by prostaglandin E2 stimulation of the prostanoid EP4 receptor via coupling to Gαi and transactivation of the epidermal growth factor receptor in HCA-7 human colon cancer cells.

Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gαi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis.

Contrary effects of sphingosine-1-phosphate on expression of α-smooth muscle actin in transforming growth factor ß1-stimulated lung fibroblasts.

Transforming growth factor-ß1 (TGFß1) plays a pivotal role in fibrosis in various organs including the lung. Following pulmonary injury, TGFß1 stimulates conversion of fibroblasts to myofibroblasts that are mainly characterized by up-regulation of α-smooth muscle actin (αSMA) expression, and the resulting excess production of extracellular matrix proteins causes fibrosis with loss of alveolar function. The present study was undertaken to define the role of the sphingosine-1-phosphate (S1P) pathway in TGFß1-induced expression of αSMA in human fetal lung fibroblasts, HFL1 cells. Analysis of mRNA revealed the existence of S1P(1), S1P(2), and S1P(3) receptor mRNAs. Treatment with TGFß1 increased sphingosine kinase (SphK) activity and S1P(3) receptor mRNA at 24h after stimulation, and pharmacological data showed the involvement of sphingomyelinase, SphK, and S1P(3) receptor in the TGFß1-induced up-regulation of αSMA with and without serum. Treatment with pertussis toxin and S1P(1) receptor antagonist W146 enhanced αSMA expression by TGFß1/serum, and S1P decreased and increased αSMA levels with and without serum, respectively. TGFß1 increased cyclooxygenase-2 expression in a manner dependent on serum and the sphingomyelinase/SphK pathway, and the response was decreased by pertussis toxin. Prostaglandin E(2), formed by TGFß1/serum stimulation, decreased the TGFß1-induced expression of αSMA via EP prostanoid receptor. These data suggest that S1P formed by TGFß1 stimulation has diverse effects on the expression of αSMA, inhibition via the S1P(1) receptor-mediated and serum-dependent expression of cyclooxygenase-2 and the resulting formation of prostaglandin E(2), and stimulation via the S1P(3) receptor in a serum-independent manner.

Regulation of human monocarboxylate transporter 4 in skeletal muscle cells: the role of protein kinase C (PKC).

In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKCδ. Moreover, rottlerin, a selective PKCδ inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1α (HIF-1α) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKCδ and HIF-1α transcription factor.

Indomethacin antagonizes EP(2) prostanoid receptor activation in LS174T human colon cancer cells.

Increases in the level of cyclooxygenase (COX)-2 and prostanoids such as prostaglandin E(2) (PGE(2)) are considered biomarkers of colorectal cancer. Therefore, non-steroidal anti-inflammatory drugs (NSAID) have been used to reduce the risk of cancer development by reducing prostanoid biosynthesis as COX inhibitors. Along with their activity as COX inhibitors, NSAID have been reported to have other effects. One major NSAID, indomethacin, has been shown to have several effects independent of COX inhibition. To further examine the COX-inhibition-independent effects of indomethacin on colorectal cancer, we used human colon cancer LS174T cells, known to have express little COX-2 and have no detectable PGE(2) production. Here we show that indomethacin has a potential antagonizing effect on human EP(2) receptors. We believe this study raises the reasons to use indomethacin as a lead-compound for setting up another EP(2) receptor-specific antagonist as a relatively cost-efficient strategy for anti-cancer medication in the future.

AMP-activated protein kinase regulates the expression of monocarboxylate transporter 4 in skeletal muscle.

AIMS: The aim of this study was to determine the effect of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, on monocarboxylate transporter 4 (MCT4) expression in rat skeletal muscle and a prototypic embryonal rhabdomyosarcoma cell line (RD cells). MAIN METHODS: We examined the alteration in Glucose transporter 4 (GLUT4) and MCT4 mRNA levels by quantitative real-time PCR. Alteration in GLUT4 and MCT4 protein levels was examined by Western blotting. KEY FINDINGS: In an in vivo study, AICAR increased MCT4 mRNA and protein levels in a fiber-type specific manner. In an in vitro study, AICAR increased MCT4 mRNA and protein levels. Moreover, AICAR-induced MCT4 expression was blocked by Compound C, an AMPK inhibitor. SIGNIFICANCE: In this study, we found that AMPK activation induced expression of MCT4 in RD cells and rat skeletal muscle in a fiber-type specific manner. These results indicate the possible involvement of an AMPK-mediated pathway associated with MCT4 expression in skeletal muscle.

Regulation of monocarboxylate transporter 1 in skeletal muscle cells by intracellular signaling pathways.

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibers also use lactic acid as a respiratory fuel. Monocarboxylate transporter (MCT) 1 has been suggested to play a major role in influx of L-lactic acid for oxidation. The regulation mechanism of MCT1 was characterized utilizing rhabdomyosarcoma cells as an in vitro skeletal muscle model. The uptake of L-lactic acid via MCT1 was studied in the presence of various intracellular regulatory pathways, including pathways mediated by protein kinases A, C and G (PKA, PKC and PKG), protein tyrosine kinase (PTK), and Ca2+/calmodulin modulators. The results showed that PKG-, PTK-, and Ca2+/calmodulin-mediated regulatory pathways play no role in the regulation of L-lactic acid uptake, but a role for PKC- and PKA-mediated pathways was apparent. Uptake of L-lactic acid appeared to be stimulated by phorbol 12-myristate 13-acetate (PMA, a PKC activator) via an increase in Vmax of transport processes with no alteration in Km. In parallel, PMA treatment also resulted in an increase in the level of MCT1 expression. On the other hand, exposure to 8-Br-cAMP, a cAMP analog, and to forskolin, an adenylyl cyclase activator, resulted in a significant decrease in L-lactic acid uptake. Additionally, 8-Br-cAMP reduced Vmax but not Km values. Parallel to the decrease in Vmax of L-lactic acid uptake, the level of MCT1 expression was decreased in response to incubation with 8-Br-cAMP. These results indicate the possible involvement of a PKC- and PKA-mediated pathway associated with expression of MCT1 and lactate transport.