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PubMed2006

Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signaling pathway.

Many small mammals have the ability to enter torpor, characterized by a controlled drop in body temperature (Tb). We hypothesized that ghrelin would modulate torpor bouts, because torpor is induced by fasting in mice coincident with elevated circulating ghrelin. Female National Institutes of Health (NIH) Swiss mice were implanted with a Tb telemeter and housed at an ambient temperature (Ta) of 18 degrees C. On fasting, all mice entered a bout of torpor (minimum Tb: 23.8+/-2.0 degrees C). Peripheral ghrelin administration (100 microg) during fasting significantly deepened the bout of torpor (Tb minimum: 19.4+/-0.5 degrees C). When the arcuate nucleus (ARC) of the hypothalamus, a ghrelin receptor-rich region of the brain, was chemically ablated with monosodium glutamate (MSG), fasted mice failed to enter torpor (minimum Tb=31.6+/-0.6 degrees C). Furthermore, ghrelin administration had no effect on the Tb minimum of ARC-ablated mice (31.8+/-0.8 degrees C). Two major pathways that regulate food intake reside in the ARC, the anorexigenic alpha-melanocyte stimulating hormone (alpha-MSH) pathway and the orexigenic neuropeptide Y (NPY) signaling pathway. Both Ay mice, which have the alpha-MSH pathway blocked, and Npy-/-mice exhibited shallow, aborted torpor bouts in response to fasting (Tb minimum: 29.1+/-0.6 degrees C and 29.9+/-1.2 degrees C, respectively). Ghrelin deepened torpor in Ay mice (Tb minimum: 22.8+/-1.3 degrees C), but had no effect in Npy-/-mice (Tb minimum: 29.5+/-0.8 degrees C). Collectively, these data suggest that ghrelin's actions on torpor are mediated via NPY neurons within the ARC.

Animals↗
PubMed2004

Fus1p interacts with components of the Hog1p mitogen-activated protein kinase and Cdc42p morphogenesis signaling pathways to control cell fusion during yeast mating.

Cell fusion in the budding yeast Saccharomyces cerevisiae is a temporally and spatially regulated process that involves degradation of the septum, which is composed of cell wall material, and occurs between conjugating cells within a prezygote, followed by plasma membrane fusion. The plasma membrane protein Fus1p is known to be required for septum degradation during cell fusion, yet its role at the molecular level is not understood. We identified Sho1p, an osmosensor for the HOG MAPK pathway, as a binding partner for Fus1 in a two-hybrid screen. The Sho1p-Fus1p interaction occurs directly and is mediated through the Sho1p-SH3 domain and a proline-rich peptide ligand on the Fus1p COOH-terminal cytoplasmic region. The cell fusion defect associated with fus1Delta mutants is suppressed by a sho1Delta deletion allele, suggesting that Fus1p negatively regulates Sho1p signaling to ensure efficient cell fusion. A two-hybrid matrix containing fusion proteins and pheromone response pathway signaling molecules reveals that Fus1p may participate in a complex network of interactions. In particular, the Fus1p cytoplasmic domain interacts with Chs5p, a protein required for secretion of specialized Chs3p-containing vesicles during bud development, and chs5Delta mutants were defective in cell surface localization of Fus1p. The Fus1p cytoplasmic domain also interacts with the activated GTP-bound form of Cdc42p and the Fus1p-SH3 domain interacts with Bni1p, a yeast formin that participates in cell fusion and controls the assembly of actin cables to polarize secretion in response to Cdc42p signaling. Taken together, our results suggest that Fus1p acts as a scaffold for the assembly of a cell surface complex involved in polarized secretion of septum-degrading enzymes and inhibition of HOG pathway signaling to promote cell fusion.

Fungal Proteins↗
PubMed2006

Highly purified lipopolysaccharides from Burkholderia cepacia complex clinical isolates induce inflammatory cytokine responses via TLR4-mediated MAPK signalling pathways and activation of NFkappaB.

In cystic fibrosis (CF), bacteria of the Burkholderia cepacia complex (Bcc) can induce a fulminant inflammation with pneumonitis and sepsis. Lipopolysaccharide (LPS) may be an important virulence factor associated with this decline but little is known about the molecular pathogenesis of Bcc LPS. In this study we have investigated the inflammatory response to highly purified LPS from different Bcc clinical isolates and the cellular signalling pathways employed. The inflammatory response (TNFalpha, IL-6) was measured in human MonoMac 6 monocytes and inhibition experiments were used to investigate the Toll-like receptors and associated adaptor molecules and pathways utilized. LPS from all clinical Bcc isolates induced significant pro-inflammatory cytokines and utilized TLR4 and CD14 to mediate activation of mitogen-activated protein kinase pathways, IkappaB-alpha degradation and NFkappaB activation. However, LPS from different clinical isolates of the same clonal strain of Burkholderia cenocepacia were found to induce a varied inflammatory response. LPS from clinical isolates of Burkholderia multivorans was found to activate the inflammatory response via MyD88-independent pathways. This study suggests that LPS alone from clinical isolates of Bcc is an important virulence factor in CF and utilizes TLR4-mediated signalling pathways to induce a significant inflammatory response.

Burkholderia Infections↗
PubMed2005

Heritable defects of the human TLR signalling pathways.

Recently, three human primary immunodeficiencies associated with impaired TLR signalling were described. Anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), either X-linked recessive or autosomal dominant, is caused by hypomorphic mutations in NEMO or hypermorphic mutation in IKBA, respectively, both involved in nuclear factor-kappaB (NF-kappaB) activation. These patients present with abnormal development of ectoderm-derived structures and suffer from a broad spectrum of infectious diseases. In vitro studies of the patients' cells showed an impaired, but not abolished, NF-kappaB activation in response to a large set of stimuli, including TLR agonists. More recently, patients with autosomal recessive amorphic mutations in IRAK4 have been reported, presenting no developmental defect and a more restricted spectrum of infectious diseases, mostly caused by pyogenic encapsulated bacteria, principally, but not exclusively Gram-positive. In vitro studies carried out with these patients' cells showed a specific impairment of the Toll-interleukin-1 receptor (TIR)-interleukin-1 receptor associated kinase (IRAK) signalling pathway. NF-kappaB- and mitogen activated protein kinase (MAPK) pathways are impaired in response to all TIR agonists tested. These data, therefore, suggest that TLRs play a critical role in host defence against pyogenic bacteria, but may be dispensable or redundant for immunity to most other infectious agents in humans.

Ectodermal Dysplasia↗
PubMed2005

Regulation of IkappaBalpha expression involves both NF-kappaB and the MAP kinase signaling pathways.

IkappaBalpha is an inhibitor of the nuclear transcription factor NF-kappaB. Binding of IkappaBalpha to NF-kappaB inactivates the transcriptional activity of NF-kappaB. Expression of IkappaBalpha itself is regulated by NF-kappaB, which provides auto-regulation of this signaling pathway. Here we present a mouse model for monitoring in vivo IkappaBalpha expression by imaging IkappaBalpha-luc transgenic mice for IkappaBalpha promoter driven luciferase activity. We demonstrated a rapid and systemic induction of IkappaBalpha expression in the transgenic mice following treatment with LPS. The induction was high in liver, spleen, lung and intestine and lower in the kidney, heart and brain. The luciferase induction in the liver correlated with increased IkappaBalpha mRNA level. Pre-treatment with proteasome inhibitor bortezomib dramatically suppressed LPS-induced luciferase activity. The p38 kinase inhibitor SB203580 also showed moderate inhibition of LPS-induced luciferase activity. Analysis of IkappaBalpha mRNA in the liver tissue showed a surprising increase of the IkappaBalpha mRNA after bortezomib and SB203580 treatments, which could be due to increased IkappaBalpha mRNA stability. Our data demonstrate that regulation of IkappaBalpha expression involves both the NF-kappaB and the p38 signaling pathways. The IkappaBalpha-luc transgenic mice are useful for analyzing IkappaBalpha expression and the NF-kappaB transcriptional activity in vivo.

Journal Article↗
PubMed2001

Effects of age on elements of insulin-signaling pathway in central nervous system of rats.

Insulin resistance is known to play a pivotal role in type 2 diabetes. Senile individuals, besides being prone to insulin resistance and, consequently, to type 2 diabetes, manifest diseases of the central nervous system (CNS) that may be influenced by disturbances of insulin signaling in the brain, such as memory impairment, Parkinson disease, and Alzheimer disease. We investigated the expression and response to insulin of elements involved in the insulin-signaling pathway in the forebrain cortex and cerebellum of rats ages 1 d to 60 wk. The protein content of insulin receptors and SRC homology adaptor protein (SHC) did not change significantly along the time frame analyzed. However, insulin-induced tyrosine phosphorylation of the insulin receptor and SHC, and the association of SHC/growth factor receptor binding protein-2 (GRB2) decreased significantly from d 1 to wk 60 of life in both types of tissues. Moreover, the expression of SH protein tyrosine phosphatase-2 (SHP2), a tyrosine phosphatase involved in insulin signal transduction and regulation of the insulin signal, decreased significantly with age progression, in both the forebrain cortex and the cerebellum of rats. Thus, elements involved in the insulin-signaling pathway are regulated at the expression and/or functional level in the CNS, and this regulation may play a role in insulin resistance in the brain.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Insulin-like growth factor-I (IGF-I) receptor activation rescues UV-damaged cells through a p38 signaling pathway. Potential role of the IGF-I receptor in DNA repair.

The activated insulin-like growth factor-I receptor (IGF-IR) is implicated in mitogenesis, transformation, and anti-apoptosis. To investigate the role of the IGF-IR in protection from UV-mimetic-induced DNA damage, 4-nitroquinoline N-oxide (4-NQO) was used. In this study we show that the activation of the IGF-IR is capable of rescuing NWTb3 cells overexpressing normal IGF-IRs from 4-NQO-induced DNA damage as demonstrated by cellular proliferation assays. This action was specific for the IGF-IR since cells expressing dominant negative IGF-IRs were not rescued from 4-NQO UV-mimetic treatment. DNA damage induced by 4-NQO in NWTb3 cells was significantly decreased after IGF-IR activation as measured by comet assay. IGF-I was also able to overcome the cell cycle arrest, observed after 4-NQO treatment, thereby enhancing the ability of NWTb3 cells to enter S phase. Interestingly, the p38 mitogen-activated protein kinase pathway was shown to represent the main signaling pathway involved in the IGF-IR-mediated rescue of UV-like damaged cells. The ability of the IGF-IR to induce DNA repair was also demonstrated by infecting NWTb3 cells with UV-irradiated adenovirus. Activation of the IGF-IR resulted in enhanced beta-galactosidase reporter gene activity demonstrating repair of the damaged DNA. This study indicates a direct role of the IGF system in the rescue of damaged cells via DNA repair.

3T3 Cells↗
PubMed1994

A vitamin D analogue KH 1060 activates the protein kinase C-c-fos signalling pathway to stimulate epidermal proliferation in murine skin.

The cellular signalling pathways of a potent 20-epi-22-oxa vitamin D3 analogue (KH 1060) were examined in vivo in a hairless mouse model. Seventy two hours after a single topical application of KH 1060 a thickening of the epidermis (from 24.8 +/- 1.2 microns at 0.01 pmol/cm2 KH 1060 to 124.2 +/- 6 microns at 5 pmol/cm2 KH 1060, P < 0.001) was elicited due to epidermal hyperproliferation. This effect could be blocked by topical 2.5 mumol/cm2 sphingosine, an inhibitor of protein kinase C. Two hours after topical application of 2.5 pmol/cm2 KH 1060 a translocation of protein kinase C activity from cytoplasm to the membrane fractions was observed. Moreover, using a reverse-transcription polymerase chain reaction technique, a transient upregulation of c-fos gene expression was seen 2 hours after topical treatment with KH 1060. The expression of c-fos was dependent on protein kinase C activation, since after pretreatment with the protein kinase C blocker sphingosine, c-fos messenger RNA was not detected. These findings strongly suggest that KH 1060 stimulates epidermal growth through activation of the protein kinase C-c-fos signalling axis in vivo.

Animals↗
PubMed2003

Epidermal growth factor up-regulates expression of transforming growth factor beta receptor type II in human dermal fibroblasts by phosphoinositide 3-kinase/Akt signaling pathway: Resistance to epidermal growth factor stimulation in scleroderma fibroblasts.

OBJECTIVE: Transforming growth factor beta receptors (TGFbetaRs) are known to be expressed at high levels in several fibrotic diseases, including systemic sclerosis. In the present study, we investigated the mechanism of up-regulation of TGFbetaR expression. METHODS: The levels of expression of TGFbetaR type II (TGFbetaRII) messenger RNA (mRNA), with or without stimulation by epidermal growth factor (EGF), were evaluated by Northern blot analysis, and the protein levels were determined by immunoblotting. The transcription activity of the TGFbetaRII gene was examined with luciferase assays using the -1670/+35 TGFbetaRII promoter luciferase construct. RESULTS: EGF up-regulates the expression of TGFbetaRII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGFbetaRII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, EGF treatment did not significantly affect the TGFbetaRII mRNA half-life. EGF-mediated induction of TGFbetaRII expression was inhibited by treatment of fibroblasts with the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002, and Akt inhibitor also blocked EGF-induced expression of TGFbetaRII. In addition, EGF induced TGFbetaRII promoter activity, and this induction was significantly blocked by wortmannin, LY294002, or Akt inhibitor. Cotransfection with a dominant-negative mutant of p85 (the regulatory component of PI 3-kinase) or Akt significantly reduced the induction of TGFbetaRII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGFbetaRII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGFbetaRII but did not show further up-regulation of TGFbetaRII expression by EGF. CONCLUSION: These results indicate that EGF-mediated induction of TGFbetaRII expression occurs at the transcription level, does not require de novo protein synthesis, and involves the PI 3-kinase/Akt signaling pathway, and that abnormal activation of EGF-mediated signaling pathways, including PI 3-kinase or Akt, might play a role in the up-regulation of TGFbetaRII in scleroderma fibroblasts.

Androstadienes↗
PubMed2005

Interaction of cellular prion and stress-inducible protein 1 promotes neuritogenesis and neuroprotection by distinct signaling pathways.

Understanding the physiological function of the cellular prion (PrPc) depends on the investigation of PrPc-interacting proteins. Stress-inducible protein 1 (STI1) is a specific PrPc ligand that promotes neuroprotection of retinal neurons through cAMP-dependent protein kinase A (PKA). Here, we examined the signaling pathways and functional consequences of the PrPc interaction with STI1 in hippocampal neurons. Both PrPc and STI1 are abundantly expressed and highly colocalized in the hippocampus in situ, indicating that they can interact in vivo. Recombinant STI1 (His6-STI1) added to hippocampal cultures interacts with PrPc at the neuronal surface and elicits neuritogenesis in wild-type neurons but not in PrPc-null cells. This effect was abolished by antibodies against either PrPc or STI1 and was dependent on the STI1 domain that binds PrPc. Binding of these proteins induced the phosphorylation/activation of the mitogen-activated protein kinase, which was essential for STI1-promoted neuritogenesis. His6-STI1, but not its counterpart lacking the PrPc binding site, prevented cell death via PKA activation. These results demonstrate that two parallel effects of the PrPc-STI1 interaction, neuritogenesis and neuroprotection, are mediated by distinct signaling pathways.

Animals↗
PubMedDate not supplied

Activation of mitochondrial K(ATP) channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway.

Activation of mitochondrial K(ATP) (mitoK(ATP)) channel induces acute ischemic preconditioning (PC) against ischemic injury. The ability of this channel to elicit late PC remains unknown. The present study tests the hypothesis that stimulation of mitoK(ATP) channel induces late PC via the protein kinase C (PKC) signaling pathway. Rats were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion (I/R). In other groups, rats were pretreated with diazoxide, a specific opener of the mitoK(ATP) channel (7 mg/kg, IV), 12, 24, 48, and 72 hours before they were subjected to I/R. A maximum reduction in infarct size was observed after 24 hours (33.3+/-2.2% versus I/R group, 62.1 +/-2.4%). Pretreatment with diazoxide did not reduce the infarct size significantly after 12, 48, and 72 hours (50.2+/-4.3%, 50.5+/-4.6%, and 58.2+/-4.9%) compared with the I/R group. The protection was blocked with 5-hydroxydecanoic acid (5-HD, 5 mg/kg IV), a relatively selective mitoK(ATP) channel blocker (56.5+/-2.7%), and chelerythrine (5 mg/kg IV), an effective PKC inhibitor (57.1+/-3.4%) administered either on the first day before diazoxide pretreatment or 10 minutes before I/R on the second day. Cell necrosis was decreased by approximately 50% in the diazoxide preconditioned hearts compared with control I/R hearts. Cell death by apoptosis was also significantly decreased in diazoxide pretreated hearts (3.2%) as compared with I/R (11.3%). In conclusion, activation of mitoK(ATP) channel with diazoxide produces late PC against reperfusion injury. The effect of mitoK(ATP) channel appears to be dependent on the PKC-mediated signal pathway.

Adenosine Triphosphate↗
PubMed2005

Signal transducers and activators of transcription 3 augments the transcriptional activity of CCAAT/enhancer-binding protein alpha in granulocyte colony-stimulating factor signaling pathway.

The Janus kinase (Jak)-Stat pathway plays an essential role in cytokine signaling. Granulocyte colony-stimulating factor (G-CSF) promotes granulopoiesis and granulocytic differentiation, and Stat3 is the principle Stat protein activated by G-CSF. Upon treatment with G-CSF, the interleukin-3-dependent cell line 32D clone 3(32Dcl3) differentiates into neutrophils, and 32Dcl3 cells expressing dominant-negative Stat3 (32Dcl3/DNStat3) proliferate in G-CSF without differentiation. Gene expression profile and quantitative PCR analysis of G-CSF-stimulated cell lines revealed that the expression of C/EBPalpha was up-regulated by the activation of Stat3. In addition, activated Stat3 bound to CCAAT/enhancer-binding protein (C/EBP)alpha, leading to the enhancement of the transcription activity of C/EBPalpha. Conditional expression of C/EBPalpha in 32Dcl3/DNStat3 cells after G-CSF stimulation abolishes the G-CSF-dependent cell proliferation and induces granulocytic differentiation. Although granulocyte-specific genes, such as the G-CSF receptor, lysozyme M, and neutrophil gelatinase-associated lipocalin precursor (NGAL) are regulated by Stat3, only NGAL was induced by the restoration of C/EBPalpha after stimulation with G-CSF in 32Dcl3/DNStat3 cells. These results show that one of the major roles of Stat3 in the G-CSF signaling pathway is to augment the function of C/EBPalpha, which is essential for myeloid differentiation. Additionally, cooperation of C/EBPalpha with other Stat3-activated proteins are required for the induction of some G-CSF responsive genes including lysozyme M and the G-CSF receptor.

Acute-Phase Proteins↗
PubMed1999

The role of star in the production of an activated ligand for the EGF receptor signaling pathway.

The Star gene is a member of the EGFR signaling pathway which has diverse functions throughout Drosophila development. In order to investigate the protein distribution for Star, we have generated a polyclonal antibody. Here, we show that the Star protein is expressed perinuclearly in the early female germline and later is found in the oocyte cytoplasm. Star is expressed at low levels in other tissues. The subcellular localization of the protein has been determined when Star is overexpressed in the eye disc. Star is located in the nuclear and contiguous endoplasmic reticulum membranes. A functional assay in the wing disc demonstrates that Star expression can activate a nonprocessed membrane-bound form of the EGFR ligand Spitz and overexpression of Star in the eye disc promotes the formation of smaller Spitz proteins. Based on these results, we propose that the Star protein is likely to be involved in Spitz ligand processing.

Animals↗
PubMed2001

New family of regulators in the environmental signaling pathway which activates the general stress transcription factor sigma(B) of Bacillus subtilis.

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

Bacillus subtilis↗
PubMed1997

Two intracellular signaling pathways for the dopamine D3 receptor: opposite and synergistic interactions with cyclic AMP.

As cerebral neurons express the dopamine D1 receptor positively coupled with adenylyl cyclase, together with the D3 receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D3 receptor signaling pathways. In NG108-15 cells transfected with the human D3 receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [3H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50-75% the quinpirole-induced [3H]thymidine incorporation. This effect was maximal with 100 nM forskolin, occurred after 6-16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D3 receptor expression up to 135%, but only after 16 h and at concentrations of > 1 microM. Thus, in this cell line, the D3 receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D3 receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D3 receptor can involve both opposite and synergistic interactions with cyclic AMP.

Cell Line↗
PubMed2003

Alphavbeta3 integrin signaling pathway is involved in insulin-like growth factor I-stimulated human extravillous trophoblast cell migration.

IGF-I and -II provide paracrine and autocrine stimuli, respectively, for extravillous trophoblast (EVT) cell migration. This study examined the role of alpha(v)beta(3) integrin and its signaling pathway in IGF-I-stimulated migration. Migration assays were conducted using cultured EVT cells treated with or without IGF-I in the presence or absence of alphaIR3, Arg-Gly-Asp (RGD) hexapeptide, and antibody against alpha(v)beta(3) integrin. Morphological changes were studied using scanning electron microscopy. Colocalization of alpha(5)beta(1) alpha(v)beta(3) integrins, vinculin, focal adhesion kinase, and paxillin were determined by immuno-cytochemistry and immunoblotting. The results showed that IGF-I could stimulate EVT cell migration in a time- and dose-dependent manner and addition of alphaIR3, Arg-Gly-Asp hexapeptide, and antibody against alpha(v)beta(3) integrin attenuated the IGF-I migratory effect. Scanning electron microscopy images revealed that IGF-I promoted lamellipodia formation. Immunostaining and immunoblotting exhibited the colocalization of alpha(v)beta(3) integrin with phosphorylated focal adhesion kinase, paxillin, and vinculin at focal adhesions after IGF-I treatment. Immunoblotting demonstrated an increase in focal adhesion kinase and paxillin tyrosine phosphorylation followed by tyrosine phosphorylation of IGF-I receptor in a time- and dose-dependent manner. These findings indicated alpha(v)beta(3) integrin localization in the core of focal adhesions of EVT cells and that alpha(v)beta(3) integrin signaling pathways are activated in IGF-I-mediated migration of these cells.

Cell Movement↗
PubMed2007

Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells.

Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.

Adipocytes↗
PubMed2001

HTLV-1 cell lines differ in constitutively activated signaling pathways that can be altered by cytokine exposure.

Examination of signaling pathways used by HTLV-1-infected rabbit cell lines revealed differences between one, RH/K30, that mediates asymptomatic infection and another, RH/K34, that causes lethal experimental leukemia. Both lines are IL-2 independent; RH/K30 produces IL-4 while RH/K34 produces IL-10. Examination of the Jak/STAT (Janus kinase/signal transducer and activator of transcription) activation of the lines revealed constitutive phosphorylation of Jak1 in both STAT6 phosphorylation, not previously reported for HTLV-1 cells, was observed in RH/K30; STAT1 and STAT3 were phosphorylated in RH/K34. Treatment with cytokines altered the activation of the STAT proteins: IL-2 induced STAT5 phosphorylation in both lines. Supernatant from RH/K34 or IL-10 induced STAT3 phosphorylation in RH/K30 cells. Supernatant from RH/K30 or IL-4 induced STAT6 phosphorylation in RH/K34 cells, which could be reversed with a Jak kinase inhibitor--AG-490.

Animals↗