Autocrine Signaling Pathway Research Articles

TGF-β Inhibits Muscle Differentiation by Blocking Autocrine Signaling Pathways Initiated by IGF-II

Skeletal muscle differentiation and regeneration are regulated by interactions between exogenous hormone- and growth factor-activated signaling cascades and endogenous muscle-specific transcriptional programs. IGF-I and IGF-II can promote muscle differentiation in vitro and can enhance muscle maintenance and repair in vivo. In contrast, members of the TGF-β superfamily prominently inhibit muscle differentiation and regeneration. In this study, we have evaluated functional interactions between IGF- and TGF-β-regulated signaling pathways during skeletal muscle differentiation. In the mouse C2 muscle cell line and in human myoblasts in primary culture, addition of TGF-β1 blocked differentiation in a dose-dependent way, inhibited expression of muscle-specific mRNAs and proteins, and impaired myotube formation. TGF-β1 also diminished stimulation of IGF-II gene expression in myoblasts, decreased IGF-II secretion, and reduced IGF-I receptor activation. To test the hypothesis that TGF-β1 prevents muscle differentiation primarily by blocking IGF-II production, we examined effects of IGF analogues on TGF-β actions in myoblasts. Although both IGF-I and IGF-II restored muscle gene and protein expression, and stimulated myotube formation in the presence of TGF-β1, they did not reduce TGF-β1-stimulated signaling, as measured by no decline in phosphorylation of SMA and mothers against decapentaplegic homolog (Smad)3, or in induction of TGF-β-activated target genes, including a Smad-dependent promoter-reporter plasmid. Our results demonstrate that TGF-β disrupts an IGF-II-stimulated autocrine amplification cascade that is necessary for muscle differentiation in vitro. Because this inhibitory pathway can be overcome by exogenous IGFs, our observations point toward potential strategies to counteract disorders that reduce muscle mass and strength.

Cancer Therapy By Targeting Hypoxia-Inducible Factor-1

Tumors are invariably less well-oxygenated than the normal tissues from which they arose. Hypoxia-inducible factor-1 (HIF-1), a key transcriptional regulator, plays a central role in the adaptation of tumor cells to hypoxia by activating the transcription of genes, which regulate several biological processes including angiogenesis, cell proliferation, survival, glucose metabolism and migration. The expression, activity and stability of HIF-1 is not only induced in response to reduced oxygen availability but also modulated through PI-3K, MAPK, autocrine signaling pathways, E3 ubiquitin ligases, and other regulators. The regulators and effects of HIF-1 in cancer have intensively provided us a new clue for the HIF-1 targeting anticancer therapy. This review evaluates the HIF-1 structure, the regulation mechanisms, the functions in cancer and corresponding anticancer strategies.

Angiotensin II induces tumor progression and fibrosis in intrahepatic cholangiocarcinoma through an interaction with hepatic stellate cells

Intrahepatic cholangiocarcinoma (ICC) is characterized as a highly fatal tumor with poor prognosis because of its strong progression, early invasion, widespread metastasis and rich cancerous stroma. Although it is widely accepted that fibroblasts facilitate stromal fibrosis and tumor progression, the mechanisms of the interaction between cancer cells and activated fibroblasts have not been fully elucidated thus far. In this study, we demonstrate the presence of angiotensin II (AngII) in ICC tissues and explore the interaction between hepatic stellate cells (HSCs) and ICC cells as one of the sources of stromal fibrosis and tumor progression through the interaction of the AngII/AngII type 1 receptor (AT-1) axis. The concentrations of AngII in ICC tissues were significantly higher than those of HCC and normal liver. Two human ICC cell lines (HuCCT-1, CCKS-1) and a human HSC cell line (LI-90) expressed AT-1 mRNA and protein. The proliferative activity of ICC cells and HSCs to which AngII was added dose-dependently increased and AT-1 antagonist inhibited the proliferative effects. HSCs to which AngII was added showed a higher expression of α-smooth muscle actin (α-SMA, a marker of activated HSCs and myofibroblasts), glial fibrillary acidic protein (GFAP, a specific marker of HSCs) and collagen type I than control cells. AT-1 antagonist also inhibited the activation and transformation of HSCs stimulated by AngII. These findings suggested that locally formed AngII in ICC tissues plays a role in the proliferation and activation of ICC cells and HSCs expressing AT-1 as a growth factor in autocrine and paracrine fashions. Our mechanistic findings provide the first insight into an autocrine and paracrine AngII-initiated signaling pathway that regulates ICC proliferation and fibrosis.

Quantitative analysis of the secretome of TGF‐β signaling‐deficient mammary fibroblasts

Transforming growth factor beta (TGF-beta) is a master regulator of autocrine and paracrine signaling pathways between a tumor and its microenvironment. Decreased expression of TGF-beta type II receptor (TbetaRII) in stromal cells is associated with increased tumor metastasis and shorter patient survival. In this study, SILAC quantitative proteomics was used to identify differentially externalized proteins in the conditioned media from the mammary fibroblasts with or without intact TbetaRII. Over 1000 proteins were identified and their relative differential levels were quantified. Immunoassays were used to further validate identification and quantification of the proteomic results. Differential expression was detected for various extracellular proteins, including proteases and their inhibitors, growth factors, cytokines, and extracellular matrix proteins. CXCL10, a cytokine found to be up-regulated in the TbetaRII knockout mammary fibroblasts, is shown to directly stimulate breast tumor cell proliferation and migration. Overall, this study revealed hundreds of specific extracellular protein changes modulated by deletion of TbetaRII in mammary fibroblasts, which may play important roles in the tumor microenvironment. These results warrant further investigation into the effects of inhibiting the TGF-beta signaling pathway in fibroblasts because systemic inhibition of TGF-beta signaling pathways is being considered as a potential cancer therapy.

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Stromal Cell Biology - A Way to Understand the Evolution of Cardiovascular Diseases -

Stromal cells, composed of fibroblasts, microvascular endothelial cells, immune cells and inflammatory cells, are critical determinants of the mechanical properties and function of the heart and vasculature, and the mechanisms whereby these types of cells are activated are important to understand the progression of cardiovascular diseases. Emerging studies have suggested that the activation of autocrine and paracrine signaling pathways by stromal cell-derived growth factors, cytokines and bioactive molecules contributes to disease progression. Disruption of the stromal network will result in alterations in the geometry and function in these organs. Interventions targeting the stromal cells (eg, myofibroblasts, microvascular endothelial cells, inflammatory cells) by pharmacological agents or direct gene delivery/small interfering RNA would be potential novel therapeutic strategies to prevent/attenuate the progression of cardiovascular disorders.

Kaposi's Sarcoma-Associated Herpesvirus Glycoproteins B and K8.1 Regulate Virion Egress and Synthesis of Vascular Endothelial Growth Factor and Viral Interleukin-6 in BCBL-1 Cells

Kaposi's sarcoma-associated herpesvirus (KSHV) viral glycoproteins play important roles in the infectious life cycle and have been implicated in KSHV-associated endothelial cell transformation, angiogenesis, and KS-induced malignancies. KSHV-associated primary effusion lymphomas (PELs) secrete high levels of vascular endothelial growth factor (VEGF) and viral interleukin-6 (vIL-6) in vitro and VEGF, vIL-6, and basic-fibroblast growth factor (b-FGF) in mouse xenografts. KSHV-encoded glycoproteins B (gB) and K8.1 stimulate VEGF secretion, most likely mediated by direct or indirect binding to cell surface receptors, including the gB-specific alphaVbeta3 and alpha3beta1 integrins. In this study, the short interfering RNA (siRNA)-mediated inhibition of either gB or K8.1 transcription by anti-gB or -K8.1 siRNAs caused a substantial reduction in virion egress and a decrease in both vIL-6 and VEGF production. Similarly, the treatment of BCBL-1 cells with anti-gB or anti-K8.1 antibodies caused a substantial reduction in vIL-6 and VEGF production. Codon-optimized versions of either wild-type gB, mutant gB having the RGD amino acid motif changed to RAA, or K8.1 efficiently rescued virion egress and VEGF and vIL-6 production. These results suggest that the binding of gB via its RGD motif to integrin receptors was not responsible for the observed gB-associated regulation of VEGF and vIL-6 transcription. Conditioned medium collected from BCBL-1 cells transfected with anti-gB and anti-K8.1 siRNAs or treated with anti-gB and anti-K8.1 antibodies exhibited a significantly reduced ability to induce the formation of the capillary network of endothelial cells compared to the ability of medium from mock-infected BCBl-1 cells. Furthermore, medium obtained from BCBL-1 cells expressing smaller amounts of gB and K8.1 produced a substantial reduction in endothelial cell migration in a vertical migration assay compared to that of control medium containing wild-type levels of gB and K8.1. These results suggest a functional linkage between gB/K8.1 synthesis and VEGF/vIL-6 transcriptional regulation via paracrine and/or autocrine signaling pathways.

GNF-2, An Allosteric BCR-ABL Inhibitor, Identifies a Novel Myristoylation-Mediated Mechanism Regulating the Ability of BCR-ABL to Activate HCK and IGF-1 Signaling.

Abstract 40Pharmacologic inhibition of BCR-ABL, using clinically active ATP-competitive inhibitors imatinib, nilotinib, and dasatinib, has been used to investigate BCR-ABL kinase activated signaling pathways. However, these agents show cross reactivity with other kinases (e.g. Kit, PDGFR, SRC family members), and their multi-targeted nature complicates assigning biological effects to the inhibition of a specific kinase target. Allosteric kinase inhibitors modulate the catalytic activity of protein kinases by binding to a site distant from the active site and inducing a protein conformation that inhibits kinase activity. These agents show promise as clinical agents and may offer advantages over ATP-competitive inhibitors in studying the function of specific kinases because they exploit binding sites and regulatory mechanisms that are unique to a particular kinase. GNF-2, a mono-selective BCR-ABL inhibitor that targets wild-type BCR-ABL and many clinically relevant imatinib resistant mutants, was recently discovered and provided the first demonstration that c-ABL kinase activity could be modulated by an inhibitor that binds outside the ATP or substrate binding sites. GNF-2 binds to a myristoyl-binding pocket in the C-lobe of the c-ABL kinase domain but its mechanism of inhibiting specific BCR-ABL kinase targets remains unclear. We previously reported that BCR-ABL activates an autocrine IGF-1 pro-survival signaling pathway in CML blast crisis cells through HCK-mediated activation of STAT5b. As GNF-2 is known to inhibit STAT5b phosphorylation, and HCK myristolyation is known to regulate its cellular localization, we hypothesized that GNF-2 inhibits BCR-ABL activation of HCK by binding to the ABL myristoyl-binding pocket and blocking access to the HCK myristoyl moiety. In support of this hypothesis, we now show that GNF-2 inhibits HCK phosphorylation and IGF-1 activation, but not HCK binding to BCR-ABL. To confirm the importance of the HCK myristoyl moiety in HCK activation, we mutated the myristoyl attachment site at position 2 in HCK from glycine to alanine. The mutant G2A HCK still interacted with BCR-ABL in co-immunoprecipitation assays but showed significantly lower levels of phosphorylation compared to wild-type HCK. To confirm that the decrease in phosphorylation was not due to mislocalization of G2A HCK, we mutated the myristoylation binding pocket of BCR-ABL by changing glutamic acid at position 505 to lysine. Similar to G2A HCK, E505K BCR-ABL still interacted with HCK, but the phosphorylation levels of HCK were dramatically reduced. To confirm that the HCK myristoyl moiety directly interacted with the ABL myristoyl-binding pocket, we used fluorescent spectroscopy to measure the ability of a myristoylated peptide corresponding to the six N-terminal amino acids of HCK to displace GNF-2. The fluorescence of GNF-2 is enhanced when it associates with the myristoyl-binding pocket of ABL. Using this assay, we calculated the Kd of GNF-2 to be 180 nM. We then assayed the ability of myristolyated HCK peptide to displace GNF-2 from ABL. We calculated the IC50 of the myristolyated HCK peptide to be 25 μM when ABL was saturated with 300 nM GNF-2. Myristate showed an IC50 of 213 μM, which is ∼ 10-fold higher than the myristoylated peptide. No binding was detected between the non-myristoylated peptide and ABL. Together, our study highlights a novel acquired function resulting from the fusion of BCR to the N-terminus of ABL, which converts the myristoyl-binding pocket in ABL from a negative regulator of kinase activity to an HCK activation motif that activates downstream IGF-1 signaling. These results also reveal the mechanism of action of the mono-selective BCR-ABL inhibitor GNF-2 and highlight the ABL myristoyl-binding pocket as a therapeutic target for inhibiting BCR-ABL activity. Disclosures:No relevant conflicts of interest to declare.

POINT: IGF IS THE MAJOR PHYSIOLOGICAL REGULATOR OF MUSCLE MASS

The insulin-like growth factors (IGFs)-I and -II are homologous peptides that are structurally related to insulin. However, IGFs retain the C-peptide, enabling binding to their main signaling receptor, the IGF type 1 receptor (IGF1R). IGF-II, however, can also bind to and signal via the A isoform of

Vascular Endothelial Growth Factor as an Autocrine Survival Factor for Retinal Pigment Epithelial Cells under Oxidative Stress via the VEGF-R2/PI3K/Akt

Vascular endothelial cell growth factor (VEGF) is strongly induced by oxidative stress in retinal pigment epithelial (RPE) cells, and VEGF-A is a survival factor for various cell types. This study was conducted to determine whether the autocrine VEGF signaling pathway in RPE cells is involved in the mechanism of adaptive response to oxidative stress. ARPE-19 cells were treated with hydrogen peroxide, and cell death was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Survival analysis was performed with pretreatment of VEGF-A-neutralizing antibodies, VEGF receptor tyrosine kinase inhibitor (SU5416), or VEGF-A receptor-neutralizing antibodies (anti-VEGF-R1 and anti-VEGF-R2). The expression of VEGF-A, -R1, -R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR or Western blot analysis. Phosphorylation of VEGF-R2 was detected with immunoprecipitation and immunoblot analysis. Hydrogen peroxide-induced cell death was promoted by pretreatment with VEGF-A and anti-VEGF-R2-neutralizing antibodies, but not with anti-VEGF-R1-neutralizing antibody. Phosphorylation of VEGF-R2 in RPE cells was induced by hydrogen peroxide, and pretreatment with anti-VEGF-A-neutralizing antibody inhibited phosphorylation. Phosphorylation of Akt under oxidative stress was abrogated by pretreatment with neutralizing antibodies against either VEGF-A or SU5416. Autocrine VEGF-A enhanced RPE cell survival under oxidative stress; the autocrine VEGF-A/VEGF-R2/PI3K/Akt pathway is involved. Neutralization of VEGF-A signaling, as in eyes with age-related macular degeneration, may influence RPE cell survival.

Cell‐autonomous intracellular androgen receptor signaling drives the growth of human prostate cancer initiating cells

The lethality of prostate cancer is due to the continuous growth of cancer initiating cells (CICs) which are often stimulated by androgen receptor (AR) signaling. However, the underlying molecular mechanism(s) for such AR-mediated growth stimulation are not fully understood. Such mechanisms may involve cancer cell-dependent induction of tumor stromal cells to produce paracrine growth factors or could involve cancer cell autonomous autocrine and/or intracellular AR signaling pathways. We utilized clinical samples, animal models and a series of AR-positive human prostate cancer cell lines to evaluate AR-mediated growth stimulation of prostate CICs. The present studies document that stromal AR expression is not required for prostate cancer growth, since tumor stroma surrounding AR-positive human prostate cancer metastases (N = 127) are characteristically AR-negative. This lack of a requirement for AR expression in tumor stromal cells is also documented by the fact that human AR-positive prostate cancer cells grow equally well when xenografted in wild-type versus AR-null nude mice. AR-dependent growth stimulation was documented to involve secretion, extracellular binding, and signaling by autocrine growth factors. Orthotopic xenograft animal studies documented that the cellautonomous autocrine growth factors which stimulate prostate CIC growth are not the andromedins secreted by normal prostate stromal cells. Such cell autonomous and extracellular autocrine signaling is necessary but not sufficient for the optimal growth of prostate CICs based upon the response to anti-androgen plus/or minus preconditioned media. AR-induced growth stimulation of human prostate CICs requires AR-dependent intracellular pathways. The identification of such AR-dependent intracellular pathways offers new leads for the development of effective therapies for prostate cancer.

Nodal Signaling via an Autocrine Pathway Promotes Proliferation of Mouse Spermatogonial Stem/Progenitor Cells Through Smad2/3 andOct-4Activation

Spermatogenesis is the process that involves the division and differentiation of spermatogonial stem cells into spermatozoa. However, the autocrine molecules and signaling pathways controlling their fate remain unknown. This study was designed to identify novel growth factors and signaling pathways that regulate proliferation, differentiation, and survival of spermatogonial stem/progenitor cells. To this end, we have for the first time explored the expression, function, and signaling pathway of Nodal, a member of the transforming growth factor-beta superfamily, in mouse spermatogonial stem/progenitor cells. We demonstrate that both Nodal and its receptors are present in these cells and in a spermatogonial stem/progenitor cell line (C18-4 cells), whereas Nodal is undetected in Sertoli cells or differentiated germ cells, as assayed by reverse transcription-polymerase chain reaction, Western blots, and immunocytochemistry. Nodal promotes proliferation of spermatogonial stem/progenitor cells and C18-4 cells, whereas Nodal receptor inhibitor SB431542 blocks their propagation as shown by proliferation and bromodeoxyuridine incorporation assays. Nodal knockdown by RNA interference results in a marked increase of cell apoptosis and a reduction of cell division as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling and proliferation assays. Conversely, overexpression of Nodal leads to an increase of cell proliferation. Nodal activates Smad2/3 phosphorylation, Oct-4 transcription, cyclin D1, and cyclin E expression, whereas SB431542 completely abolishes their increase. Together, Nodal was identified as the first autocrine signaling molecule that promotes proliferation of mouse spermatogonial stem/progenitor cells via Smad2/3 and Oct-4 activation. This study thus provides novel and important insights into molecular mechanisms regulating proliferation and survival of spermatogonial stem/progenitor cells.

Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells

Hedgehog signaling is thought to play a role in several human cancers including prostate cancer. Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation. Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B). Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold. Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1). Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog. In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881. Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment. This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients with advanced prostate cancer.

Fibroblast Growth Factor (FGF) and FGF Receptor-Mediated Autocrine Signaling in Non-Small-Cell Lung Cancer Cells

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(+/-)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

Molecular Dissection of the Human α2-Macroglobulin Subunit Reveals Domains with Antagonistic Activities in Cell Signaling

alpha(2)-Macroglobulin (alpha(2)M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in alpha(2)M are responsible for its various functions, we hypothesized that the overall effects of alpha(2)M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). FP6 rapidly and robustly activated Akt and ERK/MAP kinase in Schwann cells and PC12 cells. This response was blocked by LRP-1 gene silencing or by co-incubation with the LRP-1 antagonist, receptor-associated protein. The activity of FP6 also was blocked by mutating Lys(1370) and Lys(1374), which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-beta (NGF-beta) but not FP6. In PC12 cells, FP6 promoted neurite outgrowth and expression of growth-associated protein-43, whereas FP3 antagonized the same responses when NGF-beta was added. The ability of FP6 to trigger LRP-1-dependent cell signaling in PC12 cells was reproduced by the 18-kDa RBD, isolated from plasma-purified alpha(2)M by proteolysis and chromatography. We propose that the effects of intact alpha(2)M on cell physiology reflect the degree of penetration of activities associated with different domains, such as FP3 and FP6, which may be regulated asynchronously by conformational change and by other regulatory proteins in the cellular microenvironment.

β2-Adrenergic Receptor Signaling Mediates Corneal Epithelial Wound Repair

Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing. Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair.

Effect of Intermittent Shear Stress on Mechanotransductive Signaling and Osteoblastic Differentiation of Bone Marrow Stromal Cells

Perfusion culture of osteoprogenitor cells seeded within porous scaffolds suitable for bone tissue engineering is known to enhance deposition of a bone-like extracellular matrix, and the underlying mechanism is thought to involve flow-induced activation of mechanotransductive signaling pathways. Basic studies have shown that mechanotransduction is enhanced by impulse flow and may be mediated through autocrine signaling pathways. To test this, an intermittent flow regimen (5 min on/5 min off ) that exerts impulses on adherent cells and permits accumulation of secreted factors in the cell microenvironment was compared to continuous flow for its ability to stimulate phosphorylation of ERK and p38, synthesis of prostaglandin E2 (PGE2), and expression of mRNA for collagen 1alpha1 (Col-1alpha1), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN). Studies were performed using bone marrow stromal cells cultured in osteogenic media, and parallel-plate flow chambers were used to exert a shear stress of 2.3 dyn/cm2 on cell layers. Results show that continuous flow significantly enhanced phosphorylation of ERK and p38 after 30 min relative to intermittent flow, while intermittent flow significantly increased accumulation of PGE2 in the circulating medium by 24 h relative to continuous flow. Neither continuous nor intermittent flow affected mRNA expression of Col-1alpha1 and OPN after 4 h, but when monolayers were stimulated for 24 h and then allowed to differentiate under static conditions for an additional 13 days, expression of Col-1alpha1, OPN, BSP, and OCN under continuous and intermittent flow was similar and significantly elevated relative to static controls. This study demonstrates that the variation of perfusion regimen modulates mechanotransductive signaling.

Correction: Article on Immunohistochemical Analysis of Phosphotyrosine STAT3 and EGFR Autocrine Signaling Pathways

Correction: Article on Immunohistochemical Analysis of Phosphotyrosine STAT3 and EGFR Autocrine Signaling Pathways

Immunohistochemical Analysis of Phosphotyrosine Signal Transducer and Activator of Transcription 3 and Epidermal Growth Factor Receptor Autocrine Signaling Pathways in Head and Neck Cancers and Metastatic Lymph Nodes

To determine the effect of tyrosine-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) immunoexpression on survival in two independent cohorts of patients with squamous cell carcinoma of the head and neck (SCCHN) and to evaluate pSTAT3, transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), and gastrin-releasing peptide receptor (GRPR) expression in matched tumor and lymph node metastases in one of these cohorts. EXPERIMENTAL TECHNIQUE: Immunostaining for pSTAT3, TGF-alpha, EGFR, and GRPR was done in two SCCHN cohorts (cohort 1, 61 tumors; cohort 2, 69 paired primary tumors and lymph node metastases). Semiquantitative scores derived from the product of staining intensity (scale 0-3) score and percentage of positive tumor cells were correlated with clinical outcome. Immunoexpression of pSTAT3 did not correlate with clinical outcome in either cohort (cohort 1, P = 0.914; cohort 2, P = 0.312). In cohort 2, TGF-alpha and EGFR expression in the primary tumors showed some association with decreased disease-free survival (P = 0.0306 and P = 0.0985, respectively). Both pSTAT3 and EGFR showed a correlation of expression between tumor and matched lymph node metastasis (P < 0.0001 and P = 0.0046, respectively). In addition, the expression of EGFR and GRPR in the primary tumors correlated with TGF-alpha expression in paired nodal metastases (P = 0.0043 and P = 0.0268, respectively). In the nodal metastases, TGF-alpha expression correlated with EGFR expression (P = 0.0069). In primary tumors, GRPR expression correlated with TGF-alpha and EGFR expression (P = 0.0378 and P = 0.0026, respectively). These findings support an autocrine signaling pathway involving TGF-alpha, EGFR, and pSTAT3 in metastatic SCCHN as well as transactivation of EGFR by GRPR via TGF-alpha, but fails to identify an independent prognostic role for pSTAT3 immunoexpression.

Good news for CB1 receptors: endogenous agonists are in the right place

Endocannabinoids are endogenous ligands of brain-type (CB1) and spleen-type (CB2) cannabinoid receptors. N-Arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) are prototype members of the fatty acid amides and the monoacylglycerols, two groups of endocannabinoids. Unlike CB1, CB2 receptors do not reside within 'caveolae', specialized membrane microdomains that are well-known modulators of the activity of a number of G protein-coupled receptors. In this issue of the British Journal of Pharmacology, Rimmerman and coworkers demonstrate that 2-AG is entirely localized in the caveolae of dorsal root ganglion cells, where also part of AEA (approximately 30%) can be detected. However, most of AEA (approximately 70%) was detected in non-caveolae fractions, that is where CB2 receptors are localized. The different interaction of AEA and 2-AG with membrane microdomains might have significant implications for endocannabinoid-dependent autocrine and/or retrograde-paracrine signalling pathways. It also raises an important question about the structural determinants responsible for a different localization of two apparently similar endocannabinoids within lipid bilayers.