The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells

Uterine fibroids (leiomyomas) are a major women's health problem. Currently, the standard for treatment remains hysterectomy, since no other treatment modalities can reduce both symptoms and recurrence. As leiomyomas are benign neoplasias of smooth muscle cells, we sought to understand the regulation of uterine smooth muscle cell mitogenesis by CCN5, a growth arrest-specific gene in vascular smooth muscle cells which is induced and maintained by heparin treatment. Using autologous human myometrial and leiomyoma smooth muscle cells, we demonstrate that the proliferation and motility of both cell types are inhibited by the overexpression of CCN5. Surprisingly, we show that even though CCN5 is induced by heparin in vascular smooth muscle cells, treatment with heparin does not induce CCN5 expression in human uterine smooth muscle cells. Furthermore, we examine CCN5 mRNA expression in 10 autologous pairs of human myometrial and leiomyoma tissues and determine that CCN5 is down-regulated in 100% of the leiomyoma tissues analysed when compared to their normal myometrial counterparts. Thus, our data strongly suggest that CCN5 may exert an important function in maintaining the normal uterine phenotype and that loss of the anti-proliferative protein CCN5 from normal myometrium may account, at least in part, for tumorigenesis.     

Molecular identification and localization of Trp homologues,putative calcium channels,in pregnant human uterus

The mechanisms underlying the switch from uterine quiescence to contractile activity in labour are not clearly understood. Increasing evidence suggests that pathways of myometrial calcium homeostasis, including store-operated calcium entry (SOCE), may play an important role. The molecular basis of the membrane-associated calcium channels contributing to SOCE in pregnant human myometrium is not known, but they are likely to be hetero- or homo-oligomeric assemblies of transient receptor potential channel (TrpC) proteins, encoded by the mammalian homologues of Drosophila Trp genes. This study has therefore determined Trp gene expression and also TrpC protein expression and localization in term pregnant human myometrial tissue and primary cultured human myometrial smooth muscle (HMSM) cells. RT-PCR amplified fragments of Trp1, Trp3, Trp4, Trp6 and Trp7. PCR products were 100% homologous to published human sequences. Western blot analysis detected TrpC1, TrpC3, TrpC4 and TrpC6 proteins, which were of expected size. Immunolocalization revealed TrpC1, TrpC3, TrpC4 and TrpC6 protein expression in myometrial tissue and HMSM cells. TrpC protein immunostaining in HMSM cells was distributed in a distinct reticular fashion. TrpC proteins may be candidate proteins forming SOCE channels in term pregnant human myometrium.     

Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells

It is well known that the smooth muscle of the human myometrium is a target for the steroid hormones progesterone (P4) and estrogen. Progesterone is believed to participate in the maintenance of pregnancy, while estrogen is possibly involved in the process of parturition by promoting cervical dilatation. We examined the combined effects of P4 and 17beta-estradiol (E2) on components of signalling pathways in human myometrial cells in vitro by immunoblotting. Long-term treatment of myometrial cells with a series of concentrations of P4 and E2 in combination caused a change in the phosphorylation status of p42/44 mitogen-activated protein kinase and of c-Jun N-terminal kinase (SAPK/JNK). P4 and E2 caused a decrease in protein expression of Gqalpha, Gzalpha, Gi1/2alpha and, to a lesser extent, G0alpha. The two steroids caused a decrease in the expression of the two small GSalpha isoforms. Cyclo-oxygenase-2 expression was increased by 2.5-fold after steroid treatment, while proliferating cell nuclear antigen expression levels remained unchanged. These observations show that the combination of P4 and E2 influences intracellular and membrane-bound components of signal transduction pathways in human myometrial cells. The implications of the two steroid hormones on intracellular signalling pathways in the human myometrium merits further investigation.     

Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics

In vitro model systems for studying uterine leiomyomas are limited in that human-derived leiomyoma cells grow poorly in culture compared with normal myometrial cells and begin to senesce early, at approximately passage 10 in our studies. To our knowledge, a good in vitro human-derived cell culturing system for leiomyomas does not exist. In an attempt to fill this void, we have immortalized a uterine leiomyoma cell line by inducing telomerase activity, which allows cells to bypass their normal programmed senescence. Telomerase activity was induced by infecting the target (uterine leiomyoma and normal myometrial) cells with a retroviral vector containing hTERT, the gene for the catalytic subunit of telomerase. Subsequent analysis by RT-PCR and the telomeric repeat amplification protocol assay confirmed expression of the inserted gene and induction of telomerase activity in leiomyoma and myometrial cells. Analysis of cells for estrogen receptor-alpha and progesterone receptor proteins by Western blotting showed no change in expression of these proteins between the immortalized and parental leiomyoma and myometrial cells. Both immortalized and parental myometrial and leiomyoma cells expressed the smooth muscle-specific cytoskeletal protein alpha-actin and were negative for mutant p53 protein as evidenced by immunocytochemical staining. The immortalized leiomyoma and myometrial cells showed no anchorage-independent growth, with the exception of a small subpopulation of immortalized leiomyoma cells at a higher passage that did form two to three small colonies (per 50,000 cells) in soft agar. None of the immortalized cells were tumorigenic in nude mice. In conclusion, our data show the successful insertion of the hTERT gene into leiomyoma and myometrial cells and the immortalization of these cell lines without phenotypic alteration from the parental cell types (up to 200 population doublings). These cells should help to advance research in understanding the molecular pathways involved in the conversion of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of uterine leiomyomas. Answers to these questions will undoubtedly lead to the development of more effective treatment and intervention regimens for clinical cases of uterine leiomyoma.     

The effects of inhibiting Rho-associated kinase with Y-27632 on force and intracellular calcium in human myometrium

Recent work has indicated that smooth muscle force production may be influenced by pathways not dependent upon the Ca2+-calmodulin phosphorylation of light chains. Few studies, however, have examined the importance of these pathways in intact muscles that contract phasically rather than tonically. Therefore, to determine whether the Ca2+-independent Rho-A and associated kinase (ROK) pathway can affect contractions of the intact human myometrium, we used Y-27632 to inhibit ROK. Three types of contractile activity were examined: spontaneous and those elicited by oxytocin and by depolarisation by high K+. Y-27632 decreased force significantly under all three conditions, without changing intracellular [Ca2+]. However, the effects on force were only large when the uterus was producing force tonically rather than phasically. This suggests that the Rho-A-ROK pathway may not be a potent modulator of force in the human myometrium under physiological conditions.     

Differential expression of the metalloproteinase MMP3 and the alpha5 integrin subunit in human myometrium at labour

Extensive tissue remodelling occurs in the human myometrium before, during and after parturition. The aim of this study was to investigate the expression of two tissue remodelling molecules, matrix metalloproteinase 3 (MMP3) and alpha5 integrin (ITGA5) subunit in human myometrium, during pregnancy and labour. mRNA and protein were isolated from human pregnant labouring and non-labouring myometrial tissue, and also from human primary uterine smooth muscle cells (SMCs). Semi-quantitative RT-PCR, real-time fluorescence RT-PCR and western blotting were subsequently performed to determine the expression levels of MMP3 and ITGA5 in the myometrial tissues during pregnancy and labour, and in the primary uterine SMCs. The expression of MMP3 and ITGA5 mRNA and protein are reported for the first time during pregnancy and labour in human myometrium. Furthermore, a significant increase in expression of MMP3 mRNA (41-fold, P = 0.001), and a significant decrease in ITGA5 mRNA expression (4-fold, P < 0.001) at labour, were observed. Protein expression of these two molecules was also altered at labour, pro-MMP3 protein expression significantly increased while ITGA5 protein expression decreased, with labour onset. Expression of these molecules was also observed in primary cultured human uterine SMCs. The differential expression of these two tissue remodelling molecules at labour and their detection in uterine SMCs highlights their potential importance in myometrial function during pregnancy, labour and post-partum.     

Human atherosclerotic plaque cells and leiomyoma cells. Comparison of in vitro growth characteristics.

Cells derived from human atherosclerotic plaques and from arterial media were compared with cells obtained from human leiomyomata and myometrium with respect to growth behavior in long-term cell culture. None of numerous variations in culture media, including alterations of serum concentration and source, improved the rate of cell multiplication or in vitro longevity. Both uterine cell types, but neither arterial cell type, multiplied after tissue dissociation with enzymes (elastase, collagenase, hyaluronidase). The replicative life-span of each of eight samples of arterial plaque cells was equal to or less than that of the corresponding medial cells. A similar relationship was observed for eight paired sets of leiomyoma and myometrial cells. The results indicate that, under the conditions of culture in vitro, cells of a bona fide smooth muscle tumor have a finite replicative life-span and smooth muscle cells of atherosclerotic plaques behave in a similar manner.     

Expression and regulation of prostaglandin E synthase isoforms in human myometrium with labour

Since the controversies regarding the use of non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclo-oxygenase (COX)-2 antagonists for the treatment of preterm labour (PTL), more emphasis has been placed on investigating the terminal synthases involved in the production of prostaglandins (PGs) to allow more targeted therapy in PTL. Prostaglandin E(2) (PGE(2)) is synthesized by one of three enzymes, cytosolic prostaglandin E synthase (cPGES), microsomal PGES-1 (mPGES-1) and microsomal PGES-2 (mPGES-2). We have determined (i) the immuno-localization of all three PGES enzymes in lower segment pregnant human myometrium, (ii) the expression of PGES and COX-2 mRNA expression at term and preterm gestation with and without labour and (iii) the effect of interleukin (IL)-1beta on COX-2 and PGES mRNA and protein expression in human myometrial smooth muscle (HMSM) cell cultures. We show mPGES-1 protein located predominantly in myometrial and vascular smooth muscle cells (SMCs), whilst mPGES-2 protein is largely in stromal cells surrounding the SMC and cPGES is diffusely located throughout the myometrium. Expression of mPGES-2 mRNA increased with term labour and PTL and expression of COX-2 and mPGES-1 mRNA with term labour, whereas cPGES expression did not change. IL-1beta stimulated release of PGE(2) by HMSM cells and increased COX-2 and mPGES-1 mRNA and protein expression. Thus, COX-2 expression and mPGES-1 expression are co-ordinately up-regulated in lower segment myometrium with term labour and with IL-1beta treatment in HMSM cells.     

Human uterine leiomyoma cells: binding and growth responses to epidermal growth factor, platelet-derived growth factor, and insulin

The specific binding of epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were measured in matching cultures of human leiomyoma and myometrial cells, along with the effects of these proteins on DNA and protein syntheses. Scatchard analyses of the binding data revealed that the EGF receptor sites/cell were significantly lower in leiomyoma than myometrial cultures. Two types of PDGF binding were observed when porcine PDGF was used, and one type was seen with human PDGF. By contrast to EGF, more PDGF receptor sites/cell were found in leiomyoma than myometrium but the receptor affinity was higher in the latter. Insulin binding was similar among the myometrial and leiomyoma cells. Protein synthesis was stimulated 3-fold by EGF, PDGF, or insulin in both cell types. DNA synthesis, was higher in myometrial than leiomyoma cells in the basal state and was stimulated by EGF, insulin, or PDGF. A synergistic stimulation (p less than 0.02) of DNA synthesis was observed in both myometrial and leiomyoma cells when EGF was added with insulin. The addition of PDGF with insulin caused only additive stimulation of DNA synthesis. However, the addition of EGF with PDGF caused a synergistic decrease (p less than 0.05) in DNA synthesis by myometrial but no leiomyoma cells. Cultures of human vascular smooth muscle cells obtained from umbilical veins gave results similar to those from myometrium. These findings single out the EGF receptor and EGF, or perhaps an EGF-like growth factor, and to a lesser degree PDGF, as potential regulators of uterine leiomyomata.     

Adult mouse myometrial label-retaining cells divide in response to gonadotropin stimulation

Conditional deletion of beta-catenin in the Müllerian duct mesenchyme results in a degenerative uterus characterized by replacement of the myometrial smooth muscle with adipose tissue. We hypothesized that the mouse myometrium houses somatic smooth muscle progenitor cells that are hormonally responsive and necessary for remodeling and regeneration during estrous cycling and pregnancy. We surmise that the phenotype observed in beta-catenin conditionally deleted mice is the result of dysregulation of these progenitor cells. The objective of this study was to identify the mouse myometrial smooth muscle progenitor cell and its niche, define the surface marker phenotype, and show a functional response of these cells to normal myometrial cycling. Uteri were labeled with 5-bromo-2'-deoxyuridine (BrdU) and chased for up to 14 weeks. Myometrial label-retaining cells (LRCs) were observed in the myometrium and stroma throughout the chase period. After 12 weeks, phenotypic analysis of the LRCs by immunofluorescence demonstrated that the majority of LRCs colocalized with alpha-smooth muscle actin, estrogen receptor-alpha, and beta-catenin. Flow cytometry of myometrial cells identified a myometrial Hoechst 33342 effluxing "side population" that expresses MISRII-Cre-driven YFP. Functional response of LRCs was investigated by human chorionic gonadotropin stimulation of week 12 chase mice and demonstrated sequential proliferation of LRCs in the endometrial stroma, followed by the myometrium. These results suggest that conventional myometrial regeneration and repair is executed by hormonally responsive stem or progenitor cells derived from the Müllerian duct mesenchyme. Disclosure of potential conflicts of interest is found at the end of this article.     

Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)-dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1)

Force generation in smooth muscle is driven by phosphorylation of myosin light chains (MYL), which is regulated by the equilibrium between the activities of myosin light chain kinase (MYLK) and myosin phosphatase (MYLP). MYLK is activated by Ca(2+)-calmodulin whereas MYLP is inhibited by phosphorylation of its myosin-binding subunit (MYPT1) by Ca(2+)-independent mechanisms, leading to generation of increased MYL phosphorylation and force for a given intracellular Ca(2+) concentration, a phenomenon known as 'calcium-sensitization'. The regulation of MYPT1 phosphorylation in human myometrium, which shows increasing phasic contractility at the onset of labour, has yet to be fully investigated. Here, we explore phosphorylation of MYPT1 at Thr696 and Thr853, alongside phosphorylation of MYL, in fresh human myometrial tissue and cultured myometrial cells. We report that pMYPT1 (Thr853) levels are dependent on the activity of Rho-associated kinase (ROCK), determined using the ROCK inhibitor g-H-1152 and siRNA-mediated knockdown of ROCK1/2, and are highly correlated to ppMYL (Thr18/Ser19) levels. Pharmacological inhibition of ROCK was associated with a decrease in oxytocin (OXT)-stimulated contractility of myometrial strips in vitro. Moreover, we have measured pMYPT1 and pMYL levels between and during spontaneous and OXT-stimulated phasic contractions by rapidly freezing contracting muscle, and demonstrate for the first time functional coupling between increases in pMYPT1 (Thr853), ppMYL (Thr18/Ser19) and phasic contractility that is ROCK-dependent. The combined approach of measuring contractility and phosphorylation has demonstrated that the phosphorylation of MYPT1 (Thr853) changes dynamically with each contraction and has elucidated a defined role for ROCK in regulating myometrial contractility through MYLP, providing new insights into uterine physiology which will stimulate further research into treatments for preterm labour.     

Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-gamma ligand, pioglitazone

Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-gamma (PPARgamma) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPARgamma ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids.     

Lipid-rich residual bodies in human myometrium: qualitative observations.

Uterine leiomyomata and histologically normal myometrium were examined by light and electron microscopy. All myometrial samples contained smooth muscle cells with distinctive cytoplasmic inclusions located at the nuclear poles. These were seen in only one case in routine paraffin wax sections, where they appeared as clear vacuoles, but they were more obvious in epoxy resin sections stained with toluidine blue. Staining with Oil Red O and for acid phosphatase was positive at the site of the vacuoles. In ultrathin sections, these structures possessed a single membrane and contained abundant lipid droplets, with a sparse, coarsely granular matrix. They were designated as lipid-rich residual bodies. Such organelles were all but absent from leiomyomata. Lipid-rich residual bodies with the features described here have not been studied in detail before in smooth muscle cells of the human myometrium. The possibility that they arise from the cyclical stimulation of lipid synthesis by ovarian hormones is discussed.     

Retinoids regulate genes involved in retinoic acid synthesis and transport in human myometrial and fibroid smooth muscle cells

BACKGROUND: Despite the fact that uterine fibroids are the most common benigntumors in women, their etiology is poorly understood. We havepreviously shown that multiple members of the retinoic acid(RA) pathway have altered expression in fibroids compared withnormal myometrium. The aims of the present study were: to investigateregulation of genes involved in the RA pathway in vitro; andto identify genes that can be used as markers to distinguishmyometrial and fibroid smooth muscle cells in culture. METHODS and RESULTS: We demonstrate here for the first time that differential expressionof aldehyde dehydrogenase 1 (ALDH1) between fibroids and myometriumis maintained in cell culture (without endothelial cells), andthat this gene is differentially regulated by retinoids in myometrialcompared with fibroid cells. RA and retinol also regulate expressionof ADH1, cellular retinol binding protein 1 and cellular RAbinding protein 2 in fibroid and myometrial cells. We show thatmany of the RA pathway genes tested maintain expression levelsand differences in vitro. We also identify nine genes that aredifferentially expressed between myometrium and fibroids andmaintain these differences and expression levels in culturedcells isolated from the same tissues. These genes can be usedas markers to distinguish myometrial and fibroid cells in culture. CONCLUSIONS: Based on these findings, we propose that the RA pathway hasan important and possible causative role in fibroid growth,as evidenced by the large number of genes with significantlyaltered expression in uterine fibroids that can be regulatedby RA.     

Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells

Angiogenesis, defined as the growth of new vessels from pre-existing vessels, involves microvascular rather than large vessel endothelial cells. Accordingly, microvascular endothelial cell (MEC) proliferation assays are an appropriate in-vitro model of angiogenesis. We have developed a method for the isolation and long-term culture of large numbers of MEC from the human myometrium, tissue readily available from hysterectomy specimens. Human myometrial MEC were positively selected from tissue dissociated sequentially with collagenase and trypsin using Ulex europeaus antigen-1 (UEA)-coated dynabeads. Cultured myometrial MEC displayed characteristic endothelial phenotype and function for up to 14 passages: cobblestone morphology, formed capillary-like tubes on Matrigel, expressed CD31, Factor VIII-related antigen, bound UEA lectin, incorporated 1,1'-dioctadecyl-1,3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labelled acetylated low density lipoprotein, migrated and proliferated in response to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), but not epidermal growth factor. Optimal growth of human myometrial MEC occurred in a simple medium comprising M199, 5 ng/ml bFGF, 15% human serum, 5% fetal calf serum (FCS) and heparin. Human serum was essential for growth, although there was a synergistic effect when FCS was included. Almost identical dose-response curves were obtained for bFGF- and VEGF-induced myometrial MEC proliferation in early and late passage cells. Therefore myometrial MEC are a good model for in-vitro studies of uterine angiogenesis, since they have a stable phenotype and proliferative responsiveness to VEGF and bFGF for up to 14 passages.     

Uterine Quiescence: The Role of Cyclic AMP

It is accepted that whilst hormones such as oxytocin, vasopressin and prostaglandin F2alpha induce myometrial contractions, essentially via an elevation of intracellular calcium, other ligands, such as beta-adrenoceptor agonists, calcitonin gene-related peptide, and prostaglandin E2, promote uterine quiescence via their ability to increase intracellular cyclic AMP levels. At present, the exact factors initiating human parturition remain unknown, and labour may occur due to a loss of uterine quiescence, an increase in uterine contractility, or a combination of both. Whilst many studies have aimed to understand the mechanisms underlying uterine contractility there is a relative paucity of data regarding myometrial relaxation. We have verified the presence of mRNA encoding adenylyl cyclase (AC) isoforms I, II, III, V, VI, VII, VIII and IX in both non-pregnant and pregnant human myometrium, and in isolated myometrial cells maintained in cell culture. Furthermore, by means of immunoblotting and immunocytochemistry, we have demonstrated the expression of these isoforms as membrane-associated AC proteins, and identified changes in individual AC isoform expression during gestation. These findings illustrate the diversity of potential cAMP generating pathways in human myometrium, and the complexity of the signal transduction systems underlying uterine quiescence. Experimental Physiology (2001) 86.2, 265-272.     

Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.