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.     

Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Role of urotensin II in peripheral tissue as an autocrine/paracrine growth factor

Urotensin II (UII), originally isolated from goby urophysis, has been shown to be an endogenous ligand for an orphan G-protein-coupled receptor, GPR14. Recent development of PCR quantitative method revealed that UII and UT receptor (GPR14) were expressed in a broad range of tissues and organs, including cardiovascular and renal system, and assumed to function as an autocrine/paracrine factor. UII is a potent vasoconstrictor peptide, whose potency is greater than any other vasoconstrictors thus far known. However, its physiological roles have been found to extend far beyond the regulation of vascular tone. In this review, we focused on the mitogenic action of UII and discuss its underlying cellular mechanisms and potential physiological/pathophysiological role in various human diseases.     

Vascular endothelial growth factor plays an important autocrine/paracrine role in the progression of osteoarthritis

Vascular endothelial growth factor (VEGF) plays an essential role in the angiogenesis of growing cartilage. Although VEGF expression in cartilage vanishes in normal adults, VEGF is known to be expressed in chondrocytes of osteoarthritic (OA) cartilage. As little information is available on the VEGF expression in the cartilage of OA-like lesions of the temporomandibular joint (TMJ), VEGF expression in the condylar cartilage of TMJs of rats affected with OA was examined. To evoke OA, mechanical stress was applied by forced jaw opening for 10 or 20 days. After 20 days, marked OA-like lesions were observed in the condyle. VEGF was expressed in the chondrocytes of the mature and hypertrophic cell layers of the intermediate and posterior region of the condyle. The percentage of VEGF immunopositive chondrocytes significantly increased with the period of applied mechanical stress. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining of the condylar cartilage showed significant increment of osteoclasts in the mineralized layer subjacent to the hypertrophic layer where high VEGF expression could be detected. The results suggest that VEGF plays an important role in the progression of OA.Eiji Tanaka and Junko Aoyama contributed equally to this work.     

Hypothalamic growth hormone-releasing factor (GRF) participates in the negative feedback regulation of growth hormone secretion

Effects of growth hormone (GH) excess on immunoreactive hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) were studied in rats. Hypothalamic GRF content significantly reduced after 7-day daily treatment with 160 micrograms of rat GH or after inoculation of GH-secreting rat pituitary tumors, MtT-F4 for 9 or 13 days and GH3 for 3 months. Basal and 59 mM K+-evoked release of GRF from incubated hypothalami diminished, more than the content, by 43-51% in MtT-F4 tumor- or by 67-83% in GH3 tumor-bearing rats. In contrast, there was a small but significant increase in content or release of SRIF in rats harboring the GH3 or MtT-F4 tumor, respectively. These results indicate the existence of a negative feedback loop via hypothalamic GRF as well as SRIF in control of GH secretion.     

Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons

The toxic effects of polycyclic aromatic hydrocarbons (PAH) on spermatogenic cells undergoing meiotic division were investigated in vitro. Toxicity was assayed as alterations in cell nucleus morphology and cell survival and by DNA flow cytometry. Benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) inhibited the progression of spermatocytes through meiotic division and were highly cytotoxic at concentrations higher than 1 microM. These results were obtained upon addition of a drug-metabolizing system, indicating that the seminiferous tubules lack the enzymes required for the initiation of PAH metabolism. The spindle poisons, e.g., vincristine and Colcemid, a group of direct-acting agents, affected spermatogenesis during meiotic division in a manner similar to that observed with PAH. In contrast, adriamycin did not inhibit meiotic division, although it did induce the formation of meiotic micronuclei as a result of chromosome breakage. It is concluded that low concentrations, i.e., 0.1 microM of PAH, strongly inhibit meiotic division, presumably after metabolic activation to reactive molecules functionally resembling direct-acting alkylating agents. High concentrations of PAH are cytotoxic.     

Cdc42 participates in the regulation of ADF/cofilin and retinal growth cone filopodia by brain derived neurotrophic factor

Rho family GTPases have important roles in mediating the effects of guidance cues and growth factors on the motility of neuronal growth cones. We previously showed that the neurotrophin BDNF regulates filopodial dynamics on growth cones of retinal ganglion cell axons through activation of the actin regulatory proteins ADF and cofilin by inhibiting a RhoA-dependent pathway that phosphorylates (inactivates) ADF/cofilin. The GTPase Cdc42 has also been implicated in mediating the effects of positive guidance cues. In this article we investigated whether Cdc42 is involved in the effects of BDNF on filopodial dynamics. BDNF treatment increases Cdc42 activity in retinal neurons, and neuronal incorporation of constitutively active Cdc42 mimics the increases in filopodial number and length. Furthermore, constitutively active and dominant negative Cdc42 decreased and increased, respectively, the activity of RhoA in retinal growth cones, indicating crosstalk between these GTPases in retinal growth cones. Constitutively active Cdc42 mimicked the activation of ADF/cofilin that resulted from BDNF treatment, while dominant negative Cdc42 blocked the effects of BDNF on filopodia and ADF/cofilin. The inability of dominant negative Cdc42 to block ADF/cofilin activation and stimulation of filopodial dynamics by the ROCK inhibitor Y-27632 indicate interaction between Cdc42 and RhoA occurs upstream of ROCK. Our results demonstrate crosstalk occurs between GTPases in mediating the effects of BDNF on growth cone motility, and Cdc42 activity can promote actin dynamics via activation of ADF/cofilin.     

Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes

In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.     

Fate of meiotic lamin C2 in rat spermatocytes cultured in the presence of okadaic acid

The fate of nuclear envelope proteins during the pachytene/metaphase I transition was investigated in rat spermatocytes cultured in vitro in the presence of the phosphatase inhibitor okadaic acid (OA). Under these experimental conditions lamin B1 and the lamina-associated proteins 2 (LAPs2) behave as already described in other cell types. In contrast to these results, meiotic lamin C2 appears to be degraded after addition of OA to the spermatocyte culture medium as this lamin was no longer detectable by immunofluorescence microscopy or by immunoblotting. Taking into account the peculiarities of the lamin C2 primary structure, it is tempting to speculate that degradation of this protein represents a critical step in the process of disassembly of the spermatocyte nuclear envelope. Received: 22 September 1999; in revised form: 25 October 1999 / Accepted: 29 October 1999     

Slow diffusion on the monolayer culture enhances auto/paracrine effects of Noggin in differentiation of human iPS cells induced by BMP

Auto/paracrine factors secreted from cells affect differentiation of human pluripotent stem cells (hPSCs). However, the molecular mechanisms underlying the role of secreted factors are not well known. We previously showed that pattern formation in hPSCs induced by BMP4 could be reproduced by a simple reaction-diffusion of BMP and Noggin, a cell-secreted BMP4 inhibitor. However, the amount of Noggin secreted is unknown.In this study, we measured the concentration of Noggin secreted during the differentiation of hPSCs induced by BMP4. The Noggin concentration in the supernatant before and after differentiation was constant at approximately 0.69 ng/mL, which is approximately 50–200 times less than expected in the model. To explain the difference between the experiment and model, we assumed that macromolecules such as heparan sulfate proteoglycan on the cell surface act as a diffusion barrier structure, where the diffusion slows down to 1/400. The model with the diffusion barrier structure reduced the Noggin concentration required to suppress differentiation in the static culture model. The model also qualitatively reproduced the pattern formation, in which only the upstream but not the downstream hPSCs were differentiated in a one-directional perfusion culture chamber, with a small change in the amount of secreted Noggin resulting in a large change in the differentiation position. These results suggest that the diffusion barrier on the cell surface might enhance the auto/paracrine effects on monolayer hPSC culture.     

Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes <Emphasis Type="Italic">in vitro</Emphasis>

Background  

TGF beta and its receptors are present in both germ cells and somatic cells of the male gonad. However, knock-out strategies for studying spermatogenesis regulation by TGF beta have been disappointing since TGF beta-or TGF beta receptor-null mice do not survive longer than a few weeks.     

A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes

This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of functional gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMP +/- FSH, meiotic induction was initiated after 3 h and germinal vesicle breakdown reached 60% by 6 h. When GA was added to the cultures at different times after the initiation of culture (0, 2, 3, 4 and 5 h), meiotic induction was immediately blocked. In addition, measurement of OCC coupling revealed that no reduction in coupling occurred during this induction period in the absence of GA. It is concluded that cumulus cells can secrete a positive factor, but that this is normally overridden by inhibitory influences transmitted through the gap junction pathway in intact complexes. Furthermore, upon exposure of complexes to a meiosis-inducing stimulus, a positive gap-junction-mediated signal now predominates to trigger germinal vesicle breakdown, and this signal is utilised throughout the induction period.     

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.