17Beta-estradiol potentiates the stimulatory effects of progesterone on cadherin-11 expression in cultured human endometrial stromal cells

Cadherin-11 (cad-11) is a novel member of the cadherin gene superfamily of calcium-dependent cell adhesion molecules. To date, the factors capable of regulating this cell adhesion molecule remain poorly characterized. We have recently determined that cad-11 expression in the human endometrium is tightly regulated during the menstrual cycle. The spatiotemporal expression of cad-11 in the stromal cells of the human endometrium during the menstrual cycle suggests that gonadal steroids regulate the expression of this endometrial cell adhesion molecule. In view of these observations, we have examined the ability of progestins, estrogens, and androgens, alone or in combination, to regulate cad-11 expression in isolated human endometrial stromal cells using Northern and Western blot analyses. In these studies, we have determined that progesterone, but not 17beta-estradiol or dihydrotestosterone, is capable of regulating cad-11 messenger RNA and protein expression levels in isolated endometrial stromal cells. In addition, 17beta-estradiol, but not dihydrotestosterone, was capable of potentiating the stimulatory effects of progesterone in a dose-dependent manner. Taken together, these observations suggest that both 17beta-estradiol and progesterone are required for maximal cad-11 expression in human endometrial stromal cells in vitro.     

Requirement for transglutaminase in progesterone-induced decidualization of human endometrial stromal cells

Differentiation of endometrial stromal cells (decidualization) is essential for embryo implantation and maintenance of pregnancy. By sequential complementary DNA subtractive hybridization, one of the messenger RNAs (mRNA) induced by progesterone in human endometrial stromal cells decidualized in vitro was identified as that of a tissue transglutaminase type II (TGase). TGase mRNA was induced within 6 h after the addition of progesterone to the culture, and the effect was dose dependent. Both the TGase inhibitor monodansylcadaverine and oligodeoxynucleotide complementary to the TGase mRNA inhibited the decidualization, as assessed by PRL production and morphological transformation. Expression of TGase mRNA in human decidua and endometria exposed to high levels of progesterone in vivo was demonstrated by Northern blotting and in situ hybridization. These data suggest that TGase is necessary for the decidualization of human endometrial stromal cells and that clarification of the mechanism of action of TGase will facilitate further insight into the diagnosis and treatment of infertility.     

NMDA induces NO release from primary cell cultures of human fetal cerebral cortex

In order to investigate the role of insulin-like growth factors (IGFs) in human ovulation, we evaluated the concentrations of IGF-binding proteins (IGFBPs) in human follicular fluid (FF). The concentrations of IGFBP-1 in the FFs of 15 women undergoing in vitro fertilization and embryo transfer were measured and related to those of 17beta-estradiol (E2), progesterone and androstenedione in the FFs. IGFBP-1 levels in the FFs were positively correlated with those of E2 and progesterone. No correlation was found between the IGFBP-1 and androstenedione levels in FFs. The concentrations of IGFBP-1 were significantly increased in the FFs which contained mature oocytes compared with those of immature oocytes, whereas IGFBP-3 in FFs tended to decrease as oocytes matured. It is suggested that IGFs may play important roles in human preovulatory processes, and that IGFBP-1 may be a valuable biochemical marker in the evaluation of oocytes.     

Nuclear envelope acts as a calcium barrier in C6 glioma cells

This study evaluated the expression of the corticosteroid-metabolizing enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) during in vitro decidualization of human endometrial stromal cells. The cultured stromal cells displayed both NADP(+)-dependent (type 1) and NAD(+)-dependent (type 2) 11 beta HSD activities under basal conditions. Although the cells did not respond to estradiol (E2) added alone, catalytic levels of both isoforms were enhanced by medroxyprogesterone acetate (MPA) and further enhanced by E2 plus MPA. Type I messenger RNA (mRNA) was undetected by Northern analysis of total RNA, but was evident as a 1.5-kilobase band in polyadenylated selected RNA from E2- plus MPA-treated cultures. Use of RT-PCR to augment the sensitivity of mRNA detection revealed the presence of type I mRNA as a faint band in the MPA-treated cultures and as an intense band in the E2- plus MPA-treated cultures. Thus, type I mRNA is present as a low abundance message in the cultured stromal cells whose steady state levels parallel progestin-enhanced enzyme activity. As the expression of several progestin-regulated decidualization markers is also augmented by E2, the results of the present study reveal a correlation between enhanced 11 beta HSD expression and the decidualization reaction. Time-course measurements indicated that elevated 11 beta HSD expression is an early event in the decidualization response, which precedes E2- plus MPA-enhanced PRL production by several days. Clear dose-response effects on both type 1 and type 2 11 beta HSD activities were obtained in cells incubated with 10(-8) mol/liter E2 added together with MPA at concentrations that approximated circulating progesterone levels from the luteal phase (10(-9) mol/liter) through pregnancy (10(-7) mol/liter). Corticosteroids are thought to exert toxic and teratogenic effects on the implanting embryo and could influence trophoblast invasion by regulating extracellular matrix turnover. Therefore, the novel finding that decidualization involves marked enhancement of the corticosteroid-metabolizing capacity of stromal cells suggests a mechanism by which decidual cells could affect the health and invasiveness of implanting trophoblastic cells.     

Induction of thioredoxin, a redox-active protein, by ovarian steroid hormones during growth and differentiation of endometrial stromal cells in vitro

Human thioredoxin (hTrx) is a cellular redox-active protein that catalyzes dithiol/disulfide exchange reactions, thus controlling multiple biological functions, including cell growth-promoting activity. Here we show that the expression of hTrx protein and messenger RNA was up-regulated by incubation with 17beta-estradiol (E2) in primary culture of stromal cells isolated from human endometrium. Maximal enhancement of hTrx protein and messenger RNA was observed after 6-12 h of incubation with 10-100 nM E2, and the enhancing effect was suppressed by tamoxifen, an estrogen antagonist. Release of hTrx into the culture medium was markedly augmented after 5-day exposure of E2 plus progesterone (P) accompanied by in vitro differentiation of endometrial stromal cells (decidualization). Immunocytochemical studies showed that hTrx was localized in the nucleus, nucleolus, and cytosol in the stromal cells. Strongly enhanced immunoreactivity for hTrx was observed in the E2-treated cells, whereas there was no apparent difference in the pattern of subcellular localization among the untreated and E2- and/or P-treated cells. Although 1-50 microg/ml recombinant hTrx alone did not promote endometrial stromal cell growth, epidermal growth factor-dependent mitogenesis was additively enhanced by hTrx. Our results indicate that hTrx modulates endometrial cell growth, acting as a comitogenic factor for epidermal growth factor, which is known to be a mediator of estrogen action. It is also suggested that hTrx is deeply involved in the hormonal control of the endometrium by E2 and P, playing a regulatory role in endometrial cell growth and differentiation.     

Nutrition management, nitrogen efficiency, and income over feed cost on dairy farms in Costa Rica

In a previous study, insulin-like growth factor-I (IGF-I) and a potential inhibitory binding protein, IGF binding protein-4 (IGFBP-4), were found to be expressed in a cell-specific and temporally dynamic manner in the mouse uterus during the periimplantation period. The mRNA levels of both IGF-I and IGFBP-4 rapidly increased between Days 5 and 6 of gestation and then declined after the establishment of embryo implantation. In the current study, we conducted in situ hybridization analysis on pregnant mouse uteri and deciduomata-induced mouse uteri to determine whether the presence of an embryo is required for uterine IGF-I and IGFBP-4 mRNA expression. Our data reveal that before implantation, the maternal hormones of pregnancy support IGF-I and IGFBP-4 mRNA expression. Beyond gestational Day 4, however, decidualization of the uterine stroma, either artificially induced or naturally induced by an implanting embryo, is sufficient for maintaining the expression of these two genes. Thus, the presence of specific embryo factors is not required for IGF-I and IGFBP-4 expression in the periimplantation uterus. These studies indicate that the expression of IGF-I and IGFBP-4 mRNAs may be associated with decidualization of uterine stromal cells. The restricted anatomical and temporal expression of IGF-I and IGFBP-4 mRNAs in the periimplantation uterus suggests a physiologic role for IGF-I and IGFBP-4 in the maintenance and expansion of decidualization.     

Ovarian steroid-modulated stromelysin-1 expression in human endometrial stromal and decidual cells

This study examined steroid-regulated expression of the metalloproteinase stromelysin-1 in primary human endometrial stromal and decidual cells. Immunoblot analysis using a specific polyclonal antibody against stromelysin-1 revealed that the progestin medroxyprogesterone acetate (MPA) produced a time-dependent reduction in a band at 50,000 mol wt. Although the cells were refractory to estradiol (E2) alone, E2 plus MPA further reduced the intensity of this stromelysin-1 zone. By 6 days of incubation, MPA inhibited levels of secreted stromelysin-1 by one third, and E2 plus MPA inhibited stromelysin-1 levels by two thirds compared with the control values. This differential responsiveness of the stromal cells to the two steroids is reported for several biochemical end points of decidualization. Northern analysis indicated pronounced inhibition of stromelysin-1 messenger ribonucleic acid (mRNA) by E2 plus MPA over a concentration range that simulated circulating progesterone levels of the luteal phase (10(-8) mol/L) through pregnancy (10(-6) mol/L). After suppression of stromelysin-1 expression in the stromal cell monolayers by E2 plus MPA, steroid withdrawal led to a several-fold enhancement of stromelysin-1 mRNA by 4 days and of the stromelysin-1 protein by 7 days. Given its actions in degrading several extracellular matrix components and activating other MMP zymogens, steroid withdrawal-enhanced stromelysin-1 activity could mediate a proteolytic cascade that promotes the rapid tissue destruction and vascular disruption associated with menstruation. Stromelysin-1 expression by cultured decidual cells isolated from first trimester endometrium was also reduced by MPA and synergistically reduced by E2 plus MPA. As activation of the 92-kilodalton gelatinase/type IV collagenase, a crucial mediator of trophoblast invasiveness, is stromelysin-1 dependent, reduced decidual stromelysin-1 production could help to limit trophoblast invasion.     

Localization and sex steroid regulation of androgen receptor gene expression in rhesus monkey uterus

OBJECTIVE: To characterize the cellular sites and hormonal regulation of uterine androgen receptor gene expression in the monkey. METHODS: Ovariectomized rhesus monkeys (five in each group) were treated with placebo (the control group), estradiol (E2), E2 plus progesterone, or E2 plus testosterone by sustained-release pellets administered subcutaneously. After 3 days of treatment, uteri were removed and uterine sections were analyzed by in situ hybridization for androgen receptor messenger RNA (mRNA). RESULTS: Androgen receptor mRNA was detected in endometrial stromal cells and myometrial smooth muscle cells, with lesser expression in endometrial epithelial cells. Both E2 and E2 plus progesterone treatment doubled androgen receptor mRNA levels in stromal cells (P < .01), whereas E2 plus testosterone treatment increased stromal androgen receptor mRNA levels by about five-fold (P < .001) compared with placebo treatment. In the endometrial epithelium, E2 alone did not increase androgen receptor mRNA levels significantly. However, the E2 plus progesterone and E2 plus testosterone treatments increased epithelial androgen receptor mRNA levels by 4.3 and 5 times, respectively (P = .008 and P < .002, respectively). Androgen receptor mRNA was distributed homogeneously in smooth muscle cells across the myometrium. Estradiol treatment alone did not increase myometrial androgen receptor mRNA levels significantly, but the E2 plus progesterone and E2 plus testosterone treatments increased myometrial androgen receptor mRNA levels by 1.8 and 2 times, respectively (P = .001 and P < .001, respectively). CONCLUSION: Androgen receptor gene expression was detected in all uterine cell compartments where it was subject to significant sex steroid regulation. The fact that androgen receptor mRNA levels were consistently up-regulated by a combined E2 plus testosterone treatment while E2 treatment alone had little or no effect shows that a collaborative action of E2 and testosterone enhances androgen receptor expression in the monkey uterus.     

Transforming growth factor-beta inhibits progesterone-induced enkephalinase expression in human endometrial stromal cells

The specific activity of enkephalinase in endometrial tissue of nonpregnant ovulatory women is correlated in a highly significant, positive manner with the plasma level of progesterone. The specific activity and levels of enkephalinase messenger ribonucleic acid and immunoreactive protein also are increased in human endometrial stromal cells in culture by treatment with a synthetic progestin, medroxyprogesterone acetate (MPA), in a time- and dose-dependent manner. From an analysis of the temporal relationship between the specific activity and half-life of enkephalinase in endometrial tissue and the level of progesterone in plasma, it appeared highly likely that some mechanism, in addition to progesterone withdrawal, was operative to reduce enkephalinase activity in endometrium during the late luteal phase of the ovarian cycle before progesterone levels had declined below those known to be effective for progesterone action. In stromal cells previously (and concurrently) treated with MPA (10(-9) mol/L), the addition of transforming growth factor-beta 1 (TGF beta 1) or TGF beta 2 (1 ng/mL) to the medium caused a decrease in enkephalinase specific activity despite the continued presence of MPA. The half-life of enkephalinase (activity) in stromal cells treated with MPA plus TGF beta 1 was 2.8 days, which is similar to the computed half-life for enkephalinase in endometrial tissue during the mid- to late secretory phase of the endometrial cycle (2.5 days). Simultaneous treatment of endometrial stromal cells with MPA (10(-9) mol/L) and TGF beta 1 (1 ng/ mL) prevented the progestin-induced increase in enkephalinase specific activity and immunoreactive enkephalinase protein. Thus, TGF beta acts to oppose the progesterone-induced increase in enkephalinase expression in endometrial stromal cells, even in the continued presence of MPA.     

Identification of estrogen regulated genes in Fe33 rat hepatoma cells by differential display polymerase chain reaction and their hormonal regulation in rat liver and uterus

We applied the differential display RT-PCR (ddRT-PCR) technology to identify estrogen-regulated hepatic genes in the estrogen receptor expressing rat hepatoma cell line Fe33. Three genes of known sequences were detected by the ddRT-PCR approach: IGF binding protein-1 (IGFBP-1), vitamin D-dependent calcium-binding protein (CaBP9k) and major acute phase protein (MAP). Effects of ethinyl estradiol on the mRNA levels of these genes were confirmed by "Northern-blot" analysis. If given in combination with dexamethasone and glucagon, ethinyl estradiol caused 40-, 15- and 11-fold increases in the mRNA steady state level of IGFBP-1, CaBP9k and MAP, respectively, in Fe33 cells 24 h after addition of hormone. Besides ethinyl estradiol, the partial estrogen agonist OH-tamoxifen caused dose dependent effects on expression of MAP and IGFBP-1. Estrogen regulation of the respective genes and the modulatory effects of progesterone (10 mg/animal/day) were studied in ovariectomized rats treated subcutaneously for 14 days with 1 microgram/animal/day estradiol. "Northern-blot" analysis of liver RNA revealed a 6-fold stimulation of IGFBP-1 mRNA levels in estradiol-treated compared to vehicle-treated rats and a weak but detectable increase of MAP mRNA steady state level (1.6-fold) upon estradiol administration. No effect of estradiol treatment could be monitored for CaBP9k in rat liver. Modulatory effects of progesterone on estradiol-stimulated expression in the liver could be monitored for IGFBP-1 only. In an extension of our investigation on the expression of the three genes in rat liver, we determined their expression and hormonal regulation in the uterus of the same animals. In the uterus, estradiol caused an increase in CaBP9k mRNA. In contrast, IGFBP-1 mRNA levels increased dramatically upon progesterone administration, whereas no effect of estradiol treatment could be detected. MAP mRNA levels increased only after coadministration of estradiol and progesterone. In conclusion, the ddRT-PCR proved to be a powerful method to identify estrogen-regulated genes. The study on the hormonal regulation of three genes stimulated by estrogen in Fe33 cells revealed similarities and differences in their regulation in vivo and in vitro.     

Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-tau (IFN-tau) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-tau (rbIFN-tau) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2alpha and PG E2 (PGE2) whereas progesterone increased PGF2alpha secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-tau attenuated both PGF2alpha and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2alpha, in stromal cells. Northern blot analysis showed that E2 and rbIFN-tau decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-tau increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-tau decreased PGFS mRNA in both cell types and this was associated with the increase in PGE2/PGF2alpha ratio. These results show that the regulation of PG synthesis by steroid hormones is different in endometrial epithelial and stromal cells in vitro. The attenuation of PGF2alpha secretion from epithelial cells and increased PGE2 production in stromal cells by rbIFN-tau are modulated by steroid hormones.     

Experimental animal models and complications of transluminal coronary angioplasty

Serial changes in the endometrial levels of estrogen and progesterone receptors (ER and PR) were measured in 50 women from days 2 to 14 of missed menses and correlated with the plasma concentrations of hCG, progesterone and 17 beta-estradiol. Both ER and PR of nuclei were higher than cytosolic proteins, with a shift in the ratio of nER/nPR to nPR from 4th day after missed menses. On Scatchard analysis of the cytosolic and nuclear binding proteins, two classes of proteins, corresponding to Type I and II, were found. While the increasing levels of hCG maintained luteal secretion of progesterone and 17 beta-estradiol at normal mid-luteal phase levels, a gradual increase in 17 beta estradiol from 9th day of missed menses was noted. This delicate balance between circulating levels of progesterone and 17 beta-estradiol and their nuclear receptors at early stages of pregnancy may be of significance.     

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell stromelysin-1 and prolactin expression

During in vitro decidualization of human endometrial stromal cells (HESCs), medroxyprogesterone acetate (MPA) inhibits expression of the potent extracellular matrix (ECM)-degrading protease stromelysin-1 (MMP-3), but enhances PRL expression. Consistent with its priming role in vivo, estradiol (E2) augments these effects. In the current study, immunoblot analysis revealed that coincubation with 10(-6) M RU 486 blocked the inhibition in HESC-secreted MMP-3 levels (50,000 mol wt) evoked by 10(-8) M E2 + 10(-7) M MPA. Although MPA can act as a glucocorticoid, the HESCs were refractory to 10(-7) M dexamethasone added alone or with E2. Because E2 elevates progesterone but not glucocorticoid receptor levels, MPA and RU 486 control MMP-3 expression as a progestin and antiprogestin, respectively. To study RU 486 involvement in steroid withdrawal leading to menstruation, HESCs were decidualized during 10 days incubation with E2 + MPA, and parallel cultures were kept in E2 + MPA or withdrawn to either control or RU 486-containing medium. Compared with E2 + MPA-suppressed HESCs, increases in levels of secreted MMP-3 (2.0-fold), and its 2.1-kilobase messenger RNA (10-fold) were observed in HESCs after 4 days of withdrawal to control medium, with much greater increases seen in RU 486-containing medium (10-fold protein, 100-fold messenger RNA). Previously, we showed that RU 486 up-regulated E2 + MPA-inhibited plasminogen activator expression in the cultured HESCs. Extrapolation of these in vitro observations to endometrial events following RU 486 administration suggests that coordinate enhancement of MMP-3 and plasminogen activator expression promotes proteolysis of the stromal/decidual ECM, which leads to endometrial sloughing. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy menstrual bleeding stemming from RU 486 administration. However, in contrast to the marked RU 486-initiated reversal of MMP-3 expression, RU 486 did not significantly reverse E2 + MPA-enhanced PRL secretion by the cultured HESCs. Interestingly, decidual PRL, unlike decidual MMP-3, does not appear to play a role in menstruation. Interleukin-1 beta counteracted E2 + MPA-mediated inhibition of secreted MMP-3 levels, implying that leukocyte/trophoblast-derived cytokines can modulate steroid-regulated MMP-3 expression by stromal/decidual cells during menstruation and pregnancy.     

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.     

Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell-cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell-cell contact caused evident morphological changes accompanied with tight cell-cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow-derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.     

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.