Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.     

Autocrine/paracrine actions of growth hormone in human melanoma cell lines

Melanoma is the most aggressive skin cancer. Its aggressiveness is most commonly attributed to ERK pathway mutations leading to constitutive signaling. Though initial tumor regression results from targeting this pathway, resistance often emerges. Interestingly, interrogation of the NCI-60 database indicates high growth hormone receptor (GHR) expression in melanoma cell lines. To further characterize melanoma, we tested responsiveness to human growth hormone (GH). GH treatment resulted in GHR signaling and increased invasion and migration, which was inhibited by a GHR monoclonal antibody (mAb) antagonist in WM35, SK-MEL 5, SK-MEL 28 and SK-MEL 119 cell lines. We also detected GH in the conditioned medium (CM) of human melanoma cell lines. GHR, JAK2 and STAT5 were basally phosphorylated in these cell lines, consistent with autocrine/paracrine GH production. Together, our results suggest that melanomas are enriched in GHR and produce GH that acts in an autocrine/paracrine manner. We suggest that GHR may constitute a therapeutic target in melanoma.     

Autocrine and paracrine interactions and neuroprotection in glaucoma

Retinal ganglion cells represent the output neurons of the retina. They are responsible for integrating electrical signals that originate with the photoreceptors and, via their axons that comprise the optic nerve, transmit that information to higher visual centers of the brain. The retinal ganglion cells reside on the inner surface of the retina and their axons course across the inner surface to exit at the back of the eye through a region known as the optic nerve head. Within this region, initiation of the degenerative processes associated with glaucoma are thought to occur, leading to degeneration of not only the optic nerve but also the retinal ganglion cells themselves. Studies aimed at understanding the mechanisms behind glaucoma have identified diverse cellular components and molecular events that occur in response to nerve injury. The challenge to date has been to identify and promote pro-survival events while suppressing those that support further degradation and loss of vision. Complicating this process is the fact that the cells and molecules involved can play multiple roles. An understanding of the players and their complex relationships is central to the development of a successful treatment strategy.     

Autocrine and paracrine growth regulation of human breast cancer

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.     

Autocrine and paracrine mechanisms regulating primordial germ cell proliferation

Although several mitogens and survival factors have been previously shown to act on primordial germ cells (PGCs) in culture, it is not clear whether they are responsible for controlling proliferation of PGCs in the embryo. We show here that during their migratory phase, PGCs do not express FGF-4, FGF-8, or FGF-17, but these FGFs are expressed by neighboring cells. Thus, any FGF action on migrating PGCs would appear to be through a paracrine mechanism. We found that after entering into the gonads, PGCs start to express FGF-4 and FGF-8. On this basis, we hypothesize that FGF signaling is involved in both a paracrine manner in initiating PGC proliferation during their migration and an autocrine manner in sustaining PGC proliferation after their arrival in the gonads. We then studied the role of soluble stem cell factor (SCF), which acts as a survival factor or a mitogen in culture, to determine whether it interacts with FGFs. We found that SCF has a complex effect on PGC proliferation. On one hand, soluble SCF promoted PGC proliferation synergistically with FGF in the absence of membrane-bound SCF. Conversely, soluble SCF inhibited FGF-stimulated proliferation of PGCs in the presence of membrane-bound SCF. We account for these findings in a model involving regulation of PGC proliferation, in which SCF modulates the response to FGFs.     

Autocrine, paracrine and juxtacrine signaling by EGFR ligands

Receptor and cytoplasmic protein tyrosine kinases play prominent roles in the control of a range of cellular processes during embryonic development and in the regulation of many metabolic and physiological processes in a variety of tissues and organs. The epidermal growth factor receptor (EGFR) is a well-known and versatile signal transducer that has been highly conserved during evolution. It functions in a wide range of cellular processes, including cell fate determination, proliferation, cell migration and apoptosis. The number of ligands that can activate the EGF receptor has increased during evolution. These ligands are synthesized as membrane-anchored precursor forms that are later shed by metalloproteinase-dependent cleavage to generate soluble ligands. In certain circumstances the membrane anchored isoforms as well as soluble growth factors may also act as biologically active ligands; therefore depending on the circumstances these ligands may induce juxtacrine, autocrine, paracrine and/or endocrine signaling. In this review, we discuss the different ways that EGFR ligands can activate the receptor and the possible biological implications.     

Autocrine and paracrine nitric oxide regulate attachment of human osteoclasts

Nitric oxide (NO) can reduce bone loss in chronic bone diseases. NO inhibits or kills osteoclasts, but the mechanism of action of NO in human bone turnover is not clear. To address this, we studied effects of NO on attachment and motility of human osteoclasts on mineralized and tissue culture substrates under defined conditions. Osteoclasts were differentiated in vitro from CD14 selected monocytes in RANKL and CSF-1, and characterized by cathepsin K expression, tartrate-resistant acid phosphatase (TRAP) activity, acid secretion, and lacunar resorption. Cell attachment was labeled with monoclonal antibody 23C6, specific for a binding domain of a key osteoclast attachment protein, the CD51/CD61 integrin dimer (alpha(v)beta(3)), with or without cell permeabilization. A ring of integrin attachment during bone degradation delimits an extracellular acid compartment, while alpha(v)beta(3) forms focal attachments on non-resorbable substrates. On resorbable substrate but not non-resorbable substrate, alpha(v)beta(3) labeling required cell permeabilization, in keeping with the membrane-matrix apposition that excludes large molecules and allows extracellular acidification. Acid secretion was labeled with the fluorescent weak base indicator lysotracker. NO donors, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), downmodulated acid secretion simultaneously with cytoskeletal rearrangement, with alpha(v)beta(3) redistributed to a discontinuous pattern that labeled, on bone substrate, without membrane permeabilization. These effects were reversible, and an inhibitor of NO synthesis, N(G)-monomethyl-L-arginine (l-NMMA), increased acid secretion and decreased heterogeneity of attachment structures, showing that NO is an autocrine regulator of attachment. A hydrolysis-resistant activating cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate replicated effects of NO donors, while an inhibiting analog, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer, opposed them. On tissue culture or mineralized substrates, NO or cGMP analogs directly regulated motility; after washout cells reattached and survived for days. We conclude that NO is produced by human osteoclasts and regulates acid secretion and cellular motility, in keeping with autocrine and paracrine NO regulation of the resorption cycle.     

Autocrine/paracrine action of oxytocin in pig endometrium

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F(2alpha) secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF(2alpha). Oxytocin increased (P < 0.01) PGF(2alpha) secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF(2alpha) secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF(2alpha) secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF(2alpha) secretion.     

Autocrine and paracrine effects of peptides on human pituitary cells.

In contrast to normal human pituitaries, GH-secreting adenomas cannot process in vivo ProSRIH whereas they do it in vitro. The existence of an endogenous factor able to inhibit ProSRIH processing in vivo was postulated and such a role was analyzed for GHRH. Results showed that when GH adenomas are incubated in vitro with GHRH 10(-8) M, their ProSRIH contents are decreased, percent inhibition being negatively correlated to the amount of endogenously released GHRH. When incubation is performed in the presence of GHRH antibody in order to block the effect of endogenous GHRH, Pro-SRIH content is increased. The same effects are observed on SRIH release: inhibition by GHRH, stimulation by GHRH antibody. Normal rabbit serum had no effect. It may therefore be concluded that the absence of ProSRIH maturation observed in adenomas in vivo may be the consequence of the GHRH release that is known to be higher from GH adenomas than from normal pituitaries.     

Autocrine and paracrine regulation by cytokines and growth factors in melanoma

Tumour development and progression involves the expression of oncogenes and inactivation of tumour suppressor genes, leading to the appearance of multiple malignant characteristics. Malignant melanoma cells express different growth factors and cytokines and their receptors in respective stages of tumour progression, which by autocrine and paracrine effects enable them to grow autonomously and confer competence to metastasis. Autocrine growth factors (bFGF, MGSA/GRO, IL-8 and sometimes IL-6, PDGF-A, IL-10) produced by melanoma cells stimulate proliferation of the producing cell itself, while paracrine growth factors (for example PDGF, EGF, TGF-beta, IL-1, GM-CSF, IGF-I, NGF, VEGF) modulate the microenvironment to the benefit of tumour growth and invasion. Paracrine effects include angiogenesis, stroma formation, modulation of host immune response, activation of proteolytic enzymes, adhesion or motility and metastasis formation. Some growth factors have inhibitory effects on melanocytes and early lesions (IL-1, IL-6, TGF-beta, OSM, TNF and IFN) but not on advanced stage melanomas, and in some cases they switch to autocrine stimulator (IL-6, TGF-beta). Understanding the involvement of different growth factors and cytokines in the molecular mechanism of melanoma progression will help to provide an insight into new future therapeutic approaches for melanoma.     

Autocrine and paracrine purinergic signaling in the most lethal types of cancer

Cancer comprises a collection of diseases that occur in almost any tissue and it is characterized by an abnormal and uncontrolled cell growth that results in tumor formation and propagation to other tissues, causing tissue and organ malfunction and death. Despite the undeniable improvement in cancer diagnostics and therapy, there is an urgent need for new therapeutic and preventive strategies with improved efficacy and fewer side effects. In this context, purinergic signaling emerges as an interesting candidate as a cancer biomarker or therapeutic target. There is abundant evidence that tumor cells have significant changes in the expression of purinergic receptors, which comprise the G-protein coupled P2Y and AdoR families of receptors and the ligand-gated ion channel P2X receptors. Tumor cells also exhibit changes in the expression of nucleotidases and other enzymes involved in nucleotide metabolism, and the concentrations of extracellular nucleotides are significantly higher than those observed in normal cells. In this review, we will focus on the potential role of purinergic signaling in the ten most lethal cancers (lung, breast, colorectal, liver, stomach, prostate, cervical, esophagus, pancreas, and ovary), which together are responsible for more than 5 million annual deaths.     

Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders

Recently melanogenic paracrine or autocrine cytokine networks have been discovered in vitro between melanocytes and other types of skin cells. These include endothelin (ET)-1, granulocyte macrophage colony stimulating factor, membrane-type stem cell factor (SCF) and growth-related oncogene-alpha for interactions between keratinocytes and melanocytes, and hepatocyte growth factor and soluble type SCF for interactions between fibroblasts and melanocytes. These networks are also associated with corresponding receptors expressed on melanocytes, including ET B receptor and the SCF receptor, c-KIT. Consistent with in vitro findings on the melanogenic paracrine or autocrine cytokine networks, we have found that the up- or down-regulation of such networks is intrinsically involved in vivo in the stimulation of melanocyte functions in several epidermal hyper- or hypo-pigmentary disorders. These are ET-1/ET B receptor as well as membrane type SCF/c-KIT for ultraviolet B-melanosis, granulocyte macrophage colony stimulating factor for ultraviolet A-melanosis, ET-1/ET B receptor as well as membrane type SCF for lentigo senilis, growth related oncogene-alpha for Riehl's melanosis, sphingosylphosphorylcholine for hyperpigmentation in atopic dermatitis, ET-1 for seborrhoeic keratosis, soluble type SCF as well as hepatocyte growth factor for dermatofibroma and café-au-lait macules, and c-KIT for vitiligo vulgaris. These unveiled regulatory mechanisms involved in the abnormal up- or down-regulated levels of lesional melanocyte function provide new insights into therapeutic tools utilizing blockage of responsible cytokine networks.     

Invited Review: Autocrine/paracrine IGF-I and skeletal muscle adaptation.

This brief review presents the basic premises suggesting that insulin-like growth factor I (IGF-I), functioning in an autocrine/paracrine mode, is an important mediator of skeletal muscle adaptation. Key intracellular signaling mechanisms associated with ligation of the primary IGF-I receptor are highlighted to illustrate the mechanisms by which IGF-I may promote muscle hypertrophy. In addition, a number of recent findings are presented that highlight the potential for interactions between IGF-I-related signaling pathways and intracellular signaling mechanisms activated by cytokines or hormonal systems.     

Autocrine and paracrine regulation of lymphocyte CB2 receptor expression by TGF-beta.

The marijuana-derived cannabinoid Delta(9)-tetrahydrocannabinol (THC) has been shown to be immunosuppressive. We report that THC induces the immunosuppressive cytokine TGF-beta by human peripheral blood lymphocytes (PBL). The ability of THC to stimulate TGF-beta production was blocked by the CB2 receptor specific antagonist SR144528 but not by the CB1 specific antagonist AM251. Furthermore, our data suggest that TGF-beta actively regulates lymphocyte CB2 receptor expression in an autocrine and paracrine manner. Whereas the addition of recombinant TGF-beta to PBL cultures downregulated CB2 receptor expression, anti-TGF-beta antibody treatment increased CB2 receptor expression. We conclude that one mechanism by which THC contributes to immune suppression is by stimulating an enhanced production of lymphocyte TGF-beta.     

Autocrine and paracrine promotion of cell survival and virus replication by human herpesvirus 8 chemokines

Human herpesvirus 8 (HHV-8), which is associated with the endothelial tumor Kaposi's sarcoma, encodes three CC/beta-chemokines. These are expressed early during productive (lytic) infection and are believed to be involved in immune evasion, in addition to viral pathogenesis via induction of angiogenic cytokines. Here we report that two of the HHV-8 chemokines, CCR8 agonists vCCL-1 and vCCL-2, have direct effects on endothelial survival and virus replication. The v-chemokines stimulated virus replication when added to infected cultures exogenously, and CCR8 knockdown absent v-chemokine supplementation inhibited virus production, indicative of autocrine effects of endogenously produced vCCLs. This was verified and proreplication functions of each chemokine were demonstrated via shRNA-mediated vCCL depletion. The v-chemokines inhibited expression of lytic cycle-induced proapoptotic protein Bim, RNA interference-mediated suppression of which mimicked v-chemokine proreplication functions. Our data show for the first time that the v-chemokines have direct effects on virus biology, independently of their postulated immune evasion functions, and suggest that in vivo the v-chemokines might play direct roles in Kaposi's sarcomagenesis via paracrine prosurvival signaling.     

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E2

This study was designed to investigate the effect of IL-1alpha-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E(2) (PGE(2)) secretion and the subsequent phenotypic effects of PGE(2) on epithelial cells. The effect of IL-1alpha on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1alpha for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1alpha for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1alpha in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1alpha is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1alpha resulted in a time-dependent increase in PGE(2) secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1alpha. Co-treatment of the cells with IL-1alpha and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1alpha mediated secretion of PGE(2). Treatment of T24 cells with 100 nM PGE(2) resulted in a significant elevation in cAMP generation confirming the expression of functional PGE(2) receptors. Finally, the effect of exogenous treatment with PGE(2) on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE(2) or 10 microM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis (P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE(2) or 10 microM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE(2) inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.     

Endocrine,paracrine and autocrine actions of prolactin on immune cells

The immune response is regulated by locally released factors, collectively referred to as cytokines. Data on the human immune system have convincingly demonstrated that the hormone prolactin (PRL), in addition to exerting its endocrine control on the immune system, acts as a cytokine in that it is released within the immune system and regulates the lymphocyte response by paracrine and autocrine mechanisms. Both lymphocyte and pituitary PRLs are under the control of immune factors. Synthesis of human PRL by lymphocytes is induced by T-cell stimuli, while increased release of PRL by the pituitary, observed in vivo after immune challenge, may be mediated by cytokines produced by monocyte-macrophages. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain basal immunocompetence. The effects of Cyclosporin (CsA) on IL-2 and PRL gene activation and the analysis of the intracellular signaling events downstream IL-2 and PRL receptors suggest coordinate actions of these two cytokines during T cell activation.     

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody. Similar effects of these antibodies were observed on the spontaneous in vitro IgG production by lymphoblastic B cells from six patients with hypergammaglobulinemia. Kinetic studies with SAC/IL-2-activated B cells revealed that the anti-TNF-alpha antibody must be present at the beginning of the culture to exert an effect on Ig production, whereas the anti-IL-6 antibody reduced Ig production even if added as late as day 3. This sequential action of TNF-alpha and IL-6 on B cell differentiation was reflected by different kinetics of release of these two cytokines into the supernatant of SAC/IL-2 activated B cells; TNF-alpha peaked at 24 h and IL-6 at 96 h after stimulation. In addition, it was shown that IL-6 production by in vitro-activated B cells was partially blocked by an anti-TNF-alpha antibody suggesting that TNF-alpha regulates IL-6 production in normal B cells via an autocrine pathway. We also investigated the effects of TGF-beta on TNF-alpha and IL-6 production by normal B cells. Although TGF-beta inhibited Ig production by in vitro-activated and in vivo-activated B cells, it did not inhibit the release of these cytokines from normal B cells. Furthermore, TGF-beta did not inhibit the induction of nuclear factor-IL-6 nor the expression of IL-6R on activated B cells. Thus, although the biologic effects of anti-IL-6 and TGF-beta on B cell Ig production are similar, their mechanisms of actions appear to be distinct.     

Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release

Extracellular nucleotides such as ATP and UTP are released in response to mechanical stimulation in different cell systems. It is becoming increasingly evident that ATP release plays a role in autocrine and paracrine stimulation of osteoblasts. Mechanical stimulation, as shear stress, membrane stretch or hypo-osmotic swelling, as well as oscillatory fluid flow, stimulates ATP release from different osteoblastic cell lines. Human osteoblast-like initial transfectant (HOBIT) cells release ATP in response to mechanical stimulation. In the present study, we show that HOBIT cells are activated by nanomolar levels of extracellular ATP, concentrations that can be detected under resting conditions and increase following hypotonic shock. Cell activation by hypotonic medium induced intracellular Ca2+ oscillations, and Egr-1 synthesis and DNA-binding activity. Quinacrine staining of living, resting cells revealed a granular fluorescence, typical of ATP-storing vesicles. Monensin prevented quinacrine staining and considerably inhibited hypotonic-induced ATP release. Finally, elevated levels of cytosolic Ca2+ activated massive ATP release and a dose-dependent loss of quinacrine granules. The contribution of a vesicular mechanism for ATP release is proposed to sustain paracrine osteoblast activation.