Ovarian cancer: homeobox genes, autocrine/paracrine growth, and kinase signaling

Epithelial ovarian cancer, the fourth leading cause of cancer deaths in American women, is currently classified by surgical and histologic appearance. However, the predictive value of this classification is limited. The risk of epithelial ovarian cancer increases with the number of ovulatory events. It is now thought that different ovarian tumors are derived from a single ovarian surface epithelial precursor cell with the degree and pattern of differentiation determined by combinatorial expression of homeobox genes normally involved in differentiation of the female genital tract. This aberrant differentiation occurs in association with histology-specific genomic aberrations, genomic instability, and resultant chromosomal changes, and may be triggered by prolonged abnormal or excessive exposure of surface epithelial cells to autocrine/paracrine stimulation by sex steroids and other growth factors. As the disease progresses, activation of kinase pathways and continued abnormal autocrine/paracrine stimulation contribute to genomic instability but also identify potential targets for novel therapeutic intervention.     

Intracellular signaling following myocardial stretch: an autocrine/paracrine loop

The stretch of adult papillary muscle elicits a chain of autocrine/paracrine events in which the Na(+)/H(+) exchanger (NHE-1) activation is the central step. This activation is induced by a sequential angiotensin II-endothelin (Ang II-ET) release and results in an increase in intracellular Na(+) (Na(+)(i)) without significant changes in intracellular pH. The increase in Na(+)(i) negatively shifts the reverse potential of the Na(+)/Ca(2+) exchanger (NCX) thus inducing cell Ca(2+) influx that augments myocardial contractility. This increase in force represents the mechanical counterpart of the autocrine/paracrine mechanism triggered by stretch and has been called the slow force response (SFR) to stretch.     

Reactive oxygen species alter autocrine and paracrine signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.     

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.     

ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. Calcium ions (Ca(2+)) play an important role in the differentiation and proliferation of hMSCs. We have demonstrated that spontaneous [Ca(2+)](i) oscillations occur without agonist stimulation in hMSCs. However, the precise mechanism of its generation remains unclear. In this study, we investigated the mechanism and role of spontaneous [Ca(2+)](i) oscillations in hMSCs and found that IP(3)-induced Ca(2+) release is essential for spontaneous [Ca(2+)](i) oscillations. We also found that an ATP autocrine/paracrine signaling pathway is involved in the oscillations. In this pathway, an ATP is secreted via a hemi-gap-junction channel; it stimulates the P(2)Y(1) receptors, resulting in the activation of PLC-beta to produce IP(3). We were able to pharmacologically block this pathway, and thereby to completely halt the [Ca(2+)](i) oscillations. Furthermore, we found that [Ca(2+)](i) oscillations were associated with NFAT translocation into the nucleus in undifferentiated hMSCs. Once the ATP autocrine/paracrine signaling pathway was blocked, it was not possible to detect the nuclear translocation of NFAT, indicating that the activation of NFAT is closely linked to [Ca(2+)](i) oscillations. As the hMSCs differentiated to adipocytes, the [Ca(2+)](i) oscillations disappeared and the translocation of NFAT ceased. These results provide new insight into the molecular and physiological mechanism of [Ca(2+)](i) oscillations in undifferentiated hMSCs.     

Conjugated linoleic acid induces human adipocyte delipidation: autocrine/paracrine regulation of MEK/ERK signaling by adipocytokines

Dietary conjugated linoleic acid (CLA) reduces body fat in animals and some humans. Here we show that trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, when added to cultures of stromal vascular cells containing newly differentiated human adipocytes, caused a time-dependent decrease in triglyceride content, insulin-stimulated glucose and fatty acid uptake, incorporation into lipid, and oxidation compared with controls. In parallel, gene expression of peroxisome proliferator-activated receptor-gamma and many of its downstream targets were diminished by trans-10, cis-12 CLA, whereas leptin gene expression was increased. Prior to changes in gene expression and metabolism, trans-10, cis-12 CLA caused a robust and sustained activation of mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling. Furthermore, the trans-10, cis-12 CLA-mediated activation of MEK/ERK could be attenuated by pretreatment with U0126 and pertussis toxin. In parallel, pretreatment with U0126 blocked the ability of trans-10, cis-12 CLA to alter gene expression and attenuate glucose and fatty acid uptake of the cultures. Intriguingly, the induction by CLA of MEK/ERK signaling was linked to hypersecretion of adipocytokines interleukin-6 and interleukin-8. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA decreases the triglyceride content of newly differentiated human adipocytes by inducing MEK/ERK signaling through the autocrine/paracrine actions of interleukins-6 and 8.     

Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling

The polarized morphology of radial glia allows them to functionally interconnect different layers of CNS tissues including the retina, cerebellum, and cortex. A likely mechanism involves propagation of transcellular Ca²⁺ waves which were proposed to involve purinergic signaling. Because it is not known whether ATP release is required for astroglial Ca²⁺ wave propagation we investigated this in mouse Müller cells, radial astroglia-like retinal cells in which in which waves can be induced and supported by Orai/TRPC1 (transient receptor potential isoform 1) channels. We found that depletion of endoplasmic reticulum (ER) stores triggers regenerative propagation of transcellular Ca²⁺ waves that is independent of ATP release and activation of P₂X and P₂Y receptors. Both the amplitude and kinetics of transcellular, depletion-induced waves were resistant to non-selective purinergic P₂ antagonists such as pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). Thus, store-operated calcium entry (SOCE) is itself sufficient for the initiation and subcellular propagation of calcium waves in radial glia.     

Regulation of spermatogonial stem cell differentiation by Sertoli cells-derived exosomes through paracrine and autocrine signaling

In the orchestrated environment of the testicular niche, the equilibrium between self-renewal and differentiation of spermatogonial stem cells (SSCs) is meticulously maintained, ensuring a stable stem cell reserve and robust spermatogenesis. Within this milieu, extracellular vesicles, specifically exosomes, have emerged as critical conveyors of intercellular communication. Despite their recognized significance, the implications of testicular exosomes in modulating SSC fate remain incompletely characterized. Given the fundamental support and regulatory influence of Sertoli cells (SCs) on SSCs, we were compelled to explore the role of SC-derived exosomes (SC-EXOs) in the SSC-testicular niche. Our investigation hinged on the hypothesis that SC-EXOs, secreted by SCs from the testes of 5-day-old mice—a developmental juncture marking the onset of SSC differentiation—participate in the regulation of this process. We discovered that exposure to SC-EXOs resulted in an upsurge of PLZF, MVH, and STRA8 expression in SSC cultures, concomitant with a diminution of ID4 and GFRA1 levels. Intriguingly, obstructing exosomal communication in a SC-SSC coculture system with the exosome inhibitor GW4869 attenuated SSC differentiation, suggesting that SC-EXOs may modulate this process via paracrine signaling. Further scrutiny revealed the presence of miR-493-5p within SC-EXOs, which suppresses Gdnf mRNA in SCs to indirectly restrain SSC differentiation through the modulation of GDNF expression—an indication of autocrine regulation. Collectively, our findings illuminate the complex regulatory schema by which SC-EXOs affect SSC differentiation, offering novel perspectives and laying the groundwork for future preclinical and clinical investigations.     

Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands

It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, βKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on βKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.     

Adrenomedullin is an autocrine/paracrine growth factor for rat vascular smooth muscle cells

Adrenomedullin is a potent vasodilator peptide secreted by vascular endothelial and smooth muscle cells. Adrenomedullin stimulates the proliferation of quiescent rat vascular smooth muscle cells (VSMCs) via p42/p44 ERK/MAP kinase activation. Recently, receptor-activity-modifying proteins (RAMPs) have been shown to transport calcitonin-receptor-like-receptor (CRLR) to the cell surface to present either as CGRP receptor or adrenomedullin receptor. We investigated whether adrenomedullin acts as an autocrine/paracrine growth factor for cultured rat VSMCs and whether coexpressions of RAMP isoform and CRLR may mediate p42/p44 ERK/MAP kinase activation by adrenomedullin. Adrenomedullin dose-dependently stimulated the proliferation of quiescent rat VSMCs, and this effect was inhibited by an adrenomedullin receptor antagonist, a MAP kinase kinase inhibitor and phosphatidylinositol 3-kinase inhibitors. Addition of either CGRP(8-37) or anti-adrenomedullin antibody to exponentially growing rat VSMCs inhibited the serum-induced cell proliferation, suggesting its role as an autocrine/paracrine growth factor. Cotransfection of RAMP2 or RAMP3 with CRLR into rat VSMCs potentiated activation of cAMP activity, but not of p42/p44 ERK/MAP kinase activity in response to adrenomedullin. Our results suggest that adrenomedullin is an autocrine/paracrine growth factor for rat VSMCs via p42/p44 ERK/MAP kinase and phosphatidylinositol 3-kinase pathways and that it is not mediated by human RAMP-CRLR receptors.     

MCP-1 expressed by osteoclasts stimulates osteoclastogenesis in an autocrine/paracrine manner

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in the recruitment and activation of leukocytes. Here, we describe that multinuclear osteoclast formation was significantly inhibited in cells derived from MCP-1-deficient mice. MCP-1 has been implicated in the regulation of osteoclast cell-cell fusion; however defects of multinuclear osteoclast formation in the cells from mice deficient in DC-STAMP, a seven transmembrane receptor essential for osteoclast cell-cell fusion, was not rescued by recombinant MCP-1. The lack of MCP-1 in osteoclasts resulted in a down-regulation of DC-STAMP, NFATc1, and cathepsin K, all of which were highly expressed in normal osteoclasts, suggesting that osteoclast differentiation was inhibited in MCP-1-deficient cells. MCP-1 alone did not induce osteoclastogenesis, however, the inhibition of osteoclastogenesis in MCP-1-deficient cells was restored by addition of recombinant MCP-1, indicating that osteoclastogenesis was regulated in an autocrine/paracrine manner by MCP-1 under the stimulation of RANKL in osteoclasts.     

Regulation of bone formation by adiponectin through autocrine/paracrine and endocrine pathways

Since interaction between bone and lipid metabolism has been suggested, this study investigated the regulation of bone metabolism by adiponectin, a representative adipokine, by analyzing deficient and overexpressing transgenic mice. We initially confirmed that adiponectin and its receptors were expressed in osteoblastic and osteoclastic cells, indicating that adiponectin can act on bone not only through an endocrine pathway as a hormone secreted from fat tissue, but also through an autocrine/paracrine pathway. There was no abnormality in bone mass or turnover of adiponectin-deficient (Ad-/-) mice, possibly due to an equivalent balance of the two pathways. In the culture of bone marrow cells from the Ad-/- mice, osteogenesis was decreased compared to the wild-type (WT) cell culture, indicating a positive effect of endogenous adiponectin through the autocrine/paracrine pathway. To examine the endocrine action of adiponectin, we analyzed transgenic mice overexpressing adiponectin in the liver, and found no abnormality in the bone. Addition of recombinant adiponectin in cultured osteoprogenitor cells suppressed osteogenesis, suggesting that the direct action of circulating adiponectin was negative for bone formation. In the presence of insulin, however, this suppression was blunted, and adiponectin enhanced the insulin-induced phosphorylations of the main downstream molecule insulin receptor substrate-1 and Akt. These lines of results suggest three distinct adiponectin actions on bone formation: a positive action through the autocrine/paracrine pathway by locally produced adiponectin, a negative action through the direct pathway by circulating adiponectin, and a positive action through the indirect pathway by circulating adiponectin via enhancement of the insulin signaling.     

Cooperative interaction of autocrine and paracrine mitogens for airway epithelial cells

Mitogens of the EGF family may play an important role in regulating the proliferation of airway epithelial cells (AEC). We examined the production of autocrine mitogenic activity by mouse AEC cultured from explants of tracheal tissue. DNA synthesis by growth-arrested AEC was stimulated by conditioned media from cells maintained in serum-free culture without exogenous growth factors. The mitogenic activity was blocked by a specific inhibitor of the EGF receptor tyrosine kinase. Furthermore, conditioned media from AEC contained molecular species that could compete with radiolabeled EGF in a receptor binding assay. However, mitogenic activity was not blocked by neutralizing antibodies to EGF or to transforming growth factor-, but was partly inhibited by co-incubation with heparin, suggesting that it might be due to a heparin-binding member of the EGF family. The activity was potentiated by co-incubation with IGF-1, analogous to the potentiation by IGF-1 of the mitogenic activity of EGF for AEC. Moreover, the autocrine mitogen produced by AEC exhibited cooperative interaction with the mitogenic activity in conditioned media from growth factor-deprived mouse lung fibroblasts, consistent with the hypothesis that interactions with mesenchymal cells could influence the proliferation of AEC in vivo.     

Intermedin/adrenomedullin2: an autocrine/paracrine factor in vascular homeostasis and disease

Intermedin(IMD)or adrenomedullin 2 is a novel peptide related to the calcitonin gene-related peptide(CGRP)family.Via calcitonin receptor-like receptor/receptor activity modifying proteins,the common receptor complexes of CGRP,IMD exerts a wide range of biological effects,especially regulation of cardiovascular homeostasis.Proteolytic processing of a larger IMD precursor yields a series of biologically active C-terminal fragments,IMD1–53,IMD1–47 and IMD8–47.IMD and its receptors are present in the cardiovascular system,and IMD is present at low levels in plasma.In the cardiovascular system,IMD has multiple functions such as regulation of blood pressure and cardiac function,pro-angiogenesis,endothelial barrier function protection,anti-oxidative stress,and anti-endoplasmic reticulum stress.IMD participates widely in the pathogenesis of atherosclerosis,hypertension,pulmonary arterial hypertension and vascular calcification.It is a vascular regulatory factor of homeostasis and a vital endogenous protective factor against vascular diseases.