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PubMed2004

Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms.

BACKGROUND: Bone marrow cell therapy is reported to contribute to collateral formation through cell incorporation into new or remodeling vessels. However, the possible role of a paracrine contribution to this effect is less well characterized. METHODS AND RESULTS: Murine marrow-derived stromal cells (MSCs) were purified by magnetic bead separation of cultured bone marrow. The release of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and monocyte chemoattractant protein-1 (MCP-1) was demonstrated by analysis of MSC conditioned media (MSC-CM). MSC-CM enhanced proliferation of endothelial cells and smooth muscle cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partly attenuated these effects. Balb/C mice (n=10) underwent distal femoral artery ligation, followed by adductor muscle injection of 1x10(6) MSCs 24 hours later. Compared with controls injected with media (n=10) or mature endothelial cells (n=8), distal limb perfusion improved, and mid-thigh conductance vessels increased in number and total cross-sectional area. MSC injection improved limb function and appearance, reduced the incidence of auto-amputation, and attenuated muscle atrophy and fibrosis. After injection, labeled MSCs were seen dispersed between muscle fibers but were not seen incorporated into mature collaterals. Injection of MSCs increased adductor muscle levels of bFGF and VEGF protein compared with controls. Finally, colocalization of VEGF and transplanted MSCs within adductor tissue was demonstrated. CONCLUSIONS: MSCs secrete a wide array of arteriogenic cytokines. MSCs can contribute to collateral remodeling through paracrine mechanisms.

Animals↗
PubMed2004

Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.

We recently demonstrated that marrow stromal cells (MSCs) augment collateral remodeling through release of several cytokines such as VEGF and bFGF rather than via cell incorporation into new or remodeling vessels. The present study was designed to characterize the full spectrum of cytokine genes expressed by MSCs and to further examine the role of paracrine mechanisms that underpin their therapeutic potential. Normal human MSCs were cultured under normoxic or hypoxic conditions for 72 hours. The gene expression profile of the cells was determined using Affymetrix GeneChips representing 12 000 genes. A wide array of arteriogenic cytokine genes were expressed at baseline, and several were induced >1.5-fold by hypoxic stress. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC conditioned media (MSC(CM)). MSC(CM) promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partially attenuated these effects. Similarly, MSC(CM) promoted smooth muscle cell proliferation and migration in a dose-dependent manner. Using a murine hindlimb ischemia model, murine MSC(CM) enhanced collateral flow recovery and remodeling, improved limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy compared with control media. These data indicate that paracrine signaling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for their effects.

Adult↗
PubMed2006

Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling.

In this study, we hypothesized that bone marrow stem cells (BMSCs) protect ischemic myocardium through paracrine effects that can be further augmented with preconditioning. In in vitro experiments, cell survival factors such as Akt and eNOS were significantly increased in BMSCs following anoxia. In the second series of experiments following coronary ligation in mice, left ventricles were randomly injected with the following: DMEM (G-1), BMSCs (G-2), and preconditioned BMSCs (G-3). Four days after myocardial infarction, BMSCs were observed within injured myocardium in G-2 and G-3. Apoptotic cardiomyocytes within periinfarct area were significantly reduced in G-3. Four weeks after myocardial infarction, smaller left ventricular (LV) dimension and increased LV ejection fraction were observed in G-3. Infarct area was significantly reduced in G-3. However, GFP+ cardiomyocytes were observed in low numbers within periinfarct area in G-2 and G-3. In conclusion, BMSCs secreted cell survival factors under ischemia, and they prevented apoptosis in cardiomyocytes adjacent to the infarcted area. Preconditioning of BMSCs enhanced their survival and ability to attenuate LV remodeling, which was attributable, in part, to paracrine effects.

Animals↗
PubMed2005

Autocrine and paracrine regulation of interleukin-8 expression in lung cancer cells.

We had previously demonstrated that lung cancer cells, upon contact with macrophages, could be induced to secrete angiogenic factors to promote tumor angiogenesis. In this study, we focused on the paracrine and autocrine regulation of interleukin (IL)-8 expression in sensitized lung cancer cells after interacting with macrophages. We found that the IL-8 mRNA expression in lung cancer cells significantly increased after coculture with phorbol myristate acetate-treated THP-1 cells and human primary lung macrophages. Fresh lung cancer CL1-5 cells cocultured with macrophage-sensitized lung cancer cells still had a 35% of increase in IL-8 mRNA expression. The addition of anti-inflammatory agents pyrrolidine dithiocarbamate, pentoxifylline, aspirin, and dexamethasone could completely suppress the expression of IL-8 mRNA in fresh/sensitized lung cancer cell cocultures. Human recombinant tumor necrosis factor (TNF)-alpha and IL-1alpha could induce IL-8 expression in lung cancer cells in a dose-dependent manner. Neutralization with TNF-alpha and IL-1alpha antibodies in cocultures decreased the levels of IL-8 expression in sensitized lung cancer cells. Nuclear factor-kappaB transcriptional activity was also suppressed by the same antibodies, as confirmed by a reporter gene assay and the electrophoretic mobility shift assay. Our results highly suggest that both autocrine and paracrine regulation are involved in IL-8 expression of lung cancer cells cocultured with macrophage. Also, the regulations of IL-8 expression in lung cancer cells were through the nuclear factor-kappaB pathway and modulated by TNF-alpha and IL-1alpha.

Antibodies↗
PubMed2005

Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals.

In diabetic LDLR-/- mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg(+)) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg(+) mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear beta-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg(+) mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL(+) (Wnt reporter); CMV-Msx2Tg(+) mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals.

Alkaline Phosphatase↗
PubMed2006

Hydrogen peroxide as a paracrine vascular mediator: regulation and signaling leading to dysfunction.

Numerous studies have demonstrated the ability of a variety of vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts, to produce reactive oxygen species (ROS). Until recently, major emphasis was placed on the production of superoxide anion (O(2)(-)) in the vasculature as a result of its ability to directly attenuate the biological activity of endothelium-derived nitric oxide (NO). The short half-life and radius of diffusion of O(2)(-) drastically limit the role of this ROS as an important paracrine hormone in vascular biology. On the contrary, in recent years, the O(2)(-) metabolite hydrogen peroxide (H(2)O(2)) has increasingly been viewed as an important cellular signaling agent in its own right, capable of modulating both contractile and growth-promoting pathways with more far-reaching effects. In this review, we will assess the vascular production of H(2)O(2), its regulation by endogenous scavenger systems, and its ability to activate a variety of vascular signaling pathways, thereby leading to vascular contraction and growth. This discussion will include the ability of H(2)O(2) to (i) initiate calcium flux as well as (ii) stimulate pathways leading to sensitization of contractile elements to calcium. The latter involves a variety of protein kinases that have also been strongly implicated in vascular hypertrophy. Previous intensive study has emphasized the ability of NADPH oxidase-derived O(2)(-) and H(2)O(2) to activate these pathways in cultured smooth muscle cells. However, growing evidence indicates a considerably more complex array of unique oxidase systems in the endothelium, media, and adventitia that appear to participate in these deleterious effects in a sequential and temporal manner. Taken together, these findings seem consistent with a paracrine effect of H(2)O(2) across the vascular wall.

Animals↗
PubMed2002

Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma.

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P <.001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold; P =.02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P =.002). This effect was inhibited by anti-IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.

Bone Marrow Cells↗
PubMed2004

The small GTPase Rac1 links the Kaposi sarcoma-associated herpesvirus vGPCR to cytokine secretion and paracrine neoplasia.

Kaposi sarcoma (KS) is a multifocal angioproliferative neoplasm strictly dependent on angiogenic growth factors and cytokines and invariably associated with infection by the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8). A G protein-coupled receptor encoded by KSHV (vGPCR) is able to initiate KS-like tumors when targeted to the vascular endothelium of mice. Analogous to human KS, vGPCR sarcomagenesis involves the paracrine secretion of angiogenic growth factors and proinflammatory molecules from vGPCR-expressing cells. Here we demonstrate that vGPCR up-regulates expression and secretion of critical KS cytokines by stimulating key transcription factors, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and nuclear factor of activated T cells (NFAT), through the activation of the small G protein Rac1. Inhibition of Rac1 blocked vGPCR-induced transcription and secretion of KS cytokines, including interleukin-6 (IL-6), IL-8, and growth-regulated oncogene alpha (GROalpha), in vitro and reduced vGPCR tumorigenesis in vivo. Moreover, endothelial-specific infection with the constitutively active Rac1QL induced vascular lesions in mice that were remarkably similar to early vGPCR experimental lesions. These results identify Rac1 as a key mediator of vGPCR paracrine neoplasia, suggesting that this small G protein and its downstream effectors may represent suitable therapeutic targets for the treatment of KS.

Animals↗
PubMed2004

Autocrine and paracrine calcium signaling by the CD38/NAD+/cyclic ADP-ribose system.

CD38, a multifunctional enzyme, generates two potent Ca2+-releasing signal metabolites, cyclic ADP-ribose (cADPR) and NAADP+, thereby upmodulating many important Ca2+-mediated cell functions. A topological paradox has long been recognized, as CD38 is an ectoenzyme, or an intravesicularly located enzyme in subcellular membrane vesicles, therefore apparently shielded from its substrate NAD+. Moreover, cADPR generated by CD38 should be unavailable to its target Ca2+ stores, the ryanodine receptors (RyR). We have solved this paradox by identifying some NAD+ and cADPR transmembrane transporters, whose interplay mediates a hitherto-unrecognized subcellular and intercellular trafficking of nucleotides that enhances intracellular Ca2+ ([Ca2+]i). Connexin 43 (Cx43) hemichannels mediate an equilibrative transport of NAD+ from the cytosol to the active site of CD38 (either ectocellular or intravesicular). Subsequent translocation of in situ-generated cADPR to reach the RyR is performed, (i) by CD38 itself (concentrative) or (ii) by nucleoside transporters (NT) (one equilibrative and three concentrative). Besides this autocrine mechanism, the same transporters also mediate intercellular (paracrine) trafficking. Thus, Cx43+ and CD38+ cells can provide cADPR to neighboring RyR+ parenchymal cells and enhance their [Ca2+]i levels and Ca2+-dependent functions accordingly. Examples of cADPR-responsive cells via paracrine processes include (i) smooth myocytes, (ii) 3T3 murine fibroblasts, (iii) hippocampal neurons, and (iv) human hemopoietic stem cells.

3T3 Cells↗
PubMed2003

Minireview: overview of the renin-angiotensin system--an endocrine and paracrine system.

Since the discovery of renin as a pressor substance in 1898, the renin-angiotensin (RAS) system has been extensively studied because it remains a prime candidate as a causative factor in the development and maintenance of hypertension. Indeed, some of the properties of the physiologically active component of the RAS, angiotensin II, include vasoconstriction, regulation of renal sodium and water absorption, and increasing thirst. Initially, its affect on blood pressure was thought to be mediated primarily through the classical endocrine pathway; that is, the generation of blood-borne angiotensin with actions in target tissues. More recently, however, it has become appreciated that a local autocrine or paracrine RAS may exist in a number of tissues, and that these may also play a significant role in regulating blood pressure. Some of the difficulties in studying tissue RAS stem from the limitations of pharmacology in not differentiating between RAS products made systemically from those synthesized locally. However, the development of transgenic animals with highly specific promoters to target the RAS to specific tissues provided important tools to dissect these systems. Thus, this minireview will discuss recent advances in understanding the relationship between endocrine and paracrine (tissue) RAS using transgenic models.

Animals↗
PubMed2004

Identification of a stanniocalcin paralog, stanniocalcin-2, in fish and the paracrine actions of stanniocalcin-2 in the mammalian ovary.

Stanniocalcin is a glycoprotein hormone important in the maintenance of calcium and phosphate homeostasis in fish. Two related mammalian stanniocalcin genes, STC1 and STC2, were found to be expressed in various tissues as paracrine regulators. We have demonstrated the existence of a second stanniocalcin gene in fish, designated fish STC2, with only 30% identity to fish STC1. However, phylogenetic analysis and comparison of the genomic structure of STC genes in vertebrates indicated that STC1 and STC2 genes were probably derived from a common ancestor gene. Based on the prominent expression of mammalian STC1 in the ovary, we tested STC2 expression in rat ovary and the regulation of STC2 expression by gonadotropins. Treatment of immature rats with pregnant mare serum gonadotropin increased STC2 transcripts, whereas subsequent treatment with human chorionic gonadotropin suppressed STC2 expression. Real-time PCR analyses also demonstrated that STC2 is expressed mainly in thecal layers. In situ hybridization studies also revealed that STC2 is expressed in thecal cell layers of antral and preovulatory follicles after gonadotropin stimulation. To elucidate the physiological functions of STC2, recombinant human and fish STC2 proteins were generated and found to be N-glycosylated homodimers. In cultured granulosa cells, treatment with human or fish STC2 suppressed FSH-induced progesterone, but not estradiol or cAMP, production. The STC2 suppression of progesterone production was associated with the inhibition of FSH-induced CYP11A and 3beta-hydroxysteroid dehydrogenase expression. Thus, STC2 is a functional homodimeric glycoprotein, and thecal cell-derived STC2 could play a paracrine role during follicular development.

Animals↗
PubMed1998

Transforming growth factor beta1 is a paracrine inhibitor of prolactin gene expression.

We have shown previously that rat mammotropes produce an activity that suppresses PRL gene expression by neighboring mammotropes. Here, we tested the hypothesis that this mammotrope-derived inhibitor is transforming growth factor-beta1 (TGFbeta1). To this end, we pursued a two-pronged strategy wherein we added exogenous TGFbeta1 to primary cultures of anterior pituitary cells transfected with a rat PRL-luc construct. Measurement of luciferase activity by luminometry of extracts revealed that administration of TGFbeta1, over a range of doses shown by others to be secreted by cultures of pituitary cells, caused a significant (P < 0.05) suppression of PRL gene expression. In contrast, immunoremoval of secreted TGFbeta1 led to an elevation of PRL promoter-driven reporter activity in these cultures. In a subsequent study, we repeated these experiments with a single cell model in an attempt to determine the demographics of the cellular responses. Accordingly, we transfected (via microinjection) individual mammotropes with the rat PRL-luc construct; exposed them to TGFbeta1, its neutralizing antibody, or respective controls; and then assessed PRL gene expression in "real-time" by quantification of photons emitted by the living cells after exposure to the substrate luciferin. Our results revealed that 1) TGFbeta1 inhibited PRL gene expression in all mammotrope studied; 2) only a subgroup of mammotropes (approximately 23%) was relieved of TGFbeta1 inhibition by antibody treatment; and 3) the growth factor exerted its inhibitory effect via a paracrine, as opposed to an autocrine, mechanism. These findings identify TGFbeta1 as the paracrine agent that exerts a tonic inhibitory influence over PRL gene expression in mammotropes.

Animals↗
PubMed1999

Lipocortin 1 (annexin 1): a candidate paracrine agent localized in pituitary folliculo-stellate cells.

It is now well established that lipocortin 1 (LC1) plays an important role as a mediator of early delayed glucocorticoid feedback action in the hypothalamo-hypophysial system. In both the hypothalamus and anterior pituitary gland, LC1 mimics some of the actions of glucocorticoids; moreover, glucocorticoids stimulate the synthesis of LC1 and cause the translocation of intracellular LC1 to the outer cell surface. The mechanism by which LC1 acts in these tissues is only partially understood, but may involve paracrine and/or autocrine actions. To address these possibilities we have investigated the localization of LC1 in the rat pituitary gland, using double labeling immunohistochemistry to identify the pituitary cell types that express LC1. At the light microscopic level LC1 was not detected in the endocrine cells in cryosections of the pituitary, but it was found in abundance in the surrounding folliculo-stellate (FS) cells. In the anterior and interme diate pituitary lobes, there was a near total colocalization of LC1 and S100, a specific marker of FS cells. By contrast, in the posterior pituitary gland, LC1 immunoreactivity was not colocalized with S100 which labeled most pituicytes, or with OX-42 monoclonal antibody, a marker of the microglial cells. Immunogold electron microscopy confirmed that LC1 is present in the nongranulated FS cells. LC1 im munoreactivity was also present in a mouse pituitary FS-like cell line (TtT/GF), particularly in the periphery of the cytoplasm. The localization of LC1 in the FS cells of the anterior pituitary gland defines LC1 as a new marker of the FS cell population. These results support our hypothesis that LC1 acts as one of the paracrine agents liberated by FS cells that modulate the release of pituitary hormones.

Animals↗
PubMed2004

Paracrine regulation of ovarian granulosa cell differentiation by stanniocalcin (STC) 1: mediation through specific STC1 receptors.

Stanniocalcin (STC) in fish maintains calcium and phosphate homeostasis, whereas mammalian STC1 shows a diverse tissue expression pattern with ovary exhibiting the highest level. Based on the known expression of STC1 in theca/interstitial cells of the ovary, we generated recombinant N-glycosylated STC1 protein and tested its ability to modulate granulosa cell differentiation. In cultured rat granulosa cells obtained from early antral follicles, treatment with STC1 suppressed FSH-stimulated progesterone biosynthesis with minimal effects on estradiol and cAMP production. In mature granulosa cells, treatment with STC1 also suppressed human chorionic gonadotropin-induced progesterone production. The inhibitory effect of STC1 was accompanied by a pronounced suppression of the CYP11A transcripts and the FSH induction of functional LH receptors. In addition, STC1 was found to act downstream of adenyl cyclases in suppressing progesterone biosynthesis. We also tested the regulation of STC1 gene expression by gonadotropins. Treatment with pregnant mare serum gonadotropin decreased STC1 transcript levels in theca cells of maturing follicles, whereas subsequent treatment with human chorionic gonadotropin led to sustained suppression in the corpora lutea. Using radiolabeled recombinant STC1, receptor assays showed specific STC1 binding with a high affinity to granulosa cells. Because STC1 is expressed in ovarian theca/interstitial cells, the present demonstration of receptor binding and the specific actions of STC1 in granulosa cells suggest the existence of a follicular paracrine system in which theca cell-derived STC1 dampens the gonadotropin stimulation of granulosa cell differentiation. The observed STC1 suppression of progesterone, but not estradiol, production further suggests the potential role of this paracrine hormone as a luteinization inhibitor.

Animals↗
PubMed2005

Intermedin functions as a pituitary paracrine factor regulating prolactin release.

Calcitonin, alpha- and beta-calcitonin gene-related peptides, amylin, and adrenomedullin belong to a unique group of peptide hormones important for homeostasis maintenance. We recently identified intermedin (IMD) as a novel member of the calcitonin/calcitonin gene-related peptide family expressed in the pituitary, digestive tract, and other organs of vertebrates. Real-time PCR and immunohistochemical analysis of pituitaries from rats at different stages of development showed that IMD is expressed in the intermediate lobe and select adrenocorticotrophs in the anterior lobe, suggesting that IMD could function as a paracrine factor regulating anterior pituitary hormone secretion. In support of a paracrine role for IMD in the pituitary, quantitative and in situ hybridization analyses showed the expression of IMD receptor transcripts including the calcitonin receptor-like receptor and receptor activity-modifying proteins in the pituitary. Treatment with IMD leads to a dose-dependent increase of prolactin release in cultured rat pituitary cells. In contrast, IMD treatment has negligible effects on the release of GH, FSH, or ACTH. Likewise, in vivo treatment with IMD leads to an elevation of plasma prolactin levels in conscious rats. Based on these functional characteristics, we hypothesized that IMD could represent one of the intermediate lobe-derived prolactin-releasing factors important for prolactin regulation during reproduction. In support of this hypothesis, studies of IMD expression in lactating and ovariectomized rats showed that pituitary IMD transcripts in lactating animals increased to more than 2-fold over nonlactating controls whereas ovariectomy leads to a 90% reduction of IMD expression in the pituitary. Of importance, subsequent treatment with 17beta-estradiol or diethylstilbestrol increased pituitary IMD expression in ovariectomized rats. In addition, analysis of the proximate region of the IMD gene promoter showed that the IMD gene promoter contains consensus estrogen response element sequences, and estrogen treatments up-regulate the promoter reporter activity in transfected pituitary cells. Collectively, the present study indicates that IMD represents a novel estrogen-dependent intermediate lobe-derived prolactin-releasing factor and could play important roles in the regulation of prolactin release during reproduction in females.

Adrenocorticotropic Hormone↗
PubMed1999

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.

Animals↗
PubMed2006

Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.

Activins↗
PubMed1998

[Paracrine regulation of renal hemodynamics].

There has been an intense interest in multiple interacting paracrine systems that influence renal hemodynamics. The contractile responses at different sites along the renal vascular network exhibit distinct characteristics, depending on their receptor populations or activation mechanisms. These differences in effector mechanisms have also coupled with variations in paracrine signals from adjoining endothelial and epithelial cells. In this review, we have focused on the roles of nitric oxide (NO) and adenosine in the regulation of renal microvasculature and how they interact with other vasoactive factors. Vasopressin (VP) V2 receptors as well as V1 receptors exist in renal vasculature, especially in afferent arterioles, and V2-receptor stimulation induced vasodilation. V2-receptor-mediated vasodilation was attenuated by L-NNA. In addition, Ang II did not affect the diameter of isolated rabbit afferent arterioles, but after the treatment of L-NNA, Ang II exerted a dose-dependent vasoconstriction. Thus, NO modulated the renal vascular actions of VP and Ang II. Adenosine causes vasoconstriction via the A1 receptors, which are restricted primarily to the afferent arterioles. This selective action of adenosine suggests that adenosine exerts selective control of the renal vasculature. Adenosine augmented renal vasoconstriction by NE and Ang II via the adenosine A1 receptor, and the A1 receptor antagonist significantly reduced NE- or Ang II-induced renal vasoconstriction. The plurality of these interactions indicates that while it is very important to understand the specific direct cellular actions of each individual factor, it is equally important to understand how the various interactions are orchestrated under in vivo conditions.

Adenosine↗