Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1-3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.     

RNA-driven genetic changes in bacteria and in human cells

As recently demonstrated in the yeast Saccharomyces cerevisiae model organism using synthetic RNA-containing oligonucleotides (oligos), RNA can serve as a template for DNA synthesis at the chromosomal level during the process of double-strand break (DSB) repair. Herein we show that the phenomenon of RNA-mediated DNA modification and repair is not limited to yeast cells. A tract of six ribonucleotides embedded in single-strand DNA oligos corresponding to either lagging or leading strand sequences could serve as a template to correct a defective lacZ marker gene in the chromosome of the bacterium Escherichia coli. In order to test the capacity of RNA to modify DNA in mammalian cells, we utilized DNA oligos containing an embedded tract of six ribonucleotides, as well as oligos mostly made of RNA. These oligos were designed to repair a chromosomal break generated within a copy of the green fluorescent protein (GFP) gene randomly integrated into the genome of human HEK-293 cells. We show that these RNA-containing oligos can serve as templates to repair a DSB in human cells and can introduce base changes into genomic or plasmid DNA. In both E. coli and human cells, the strand bias of chromosomal gene correction by the single-strand RNA-containing oligos was the same as that obtained for the corresponding DNA molecules. Therefore, the RNA-containing oligos are not converted into a cDNA before annealing with complementary DNA. Overall, we demonstrate that in both bacterial and human cells, as in yeast, RNA sequences can have a direct role in DNA genetic modification and remodeling.     

Dihydrotestosterone potentiates insulin to up-regulate prokineticin-1 in decidualizing human endometrial stromal cells

Prokineticin 1 (PROK1) is a key regulator of embryo implantation and placentation, and its dysregulation is associated with pregnancy complications, such as pre-eclampsia and foetal growth restriction. We have previously shown that insulin strongly enhances the expression of PROK1 in human decidualizing stromal cells. Here, we demonstrate that dihydrotestosterone (DHT), but not testosterone, potentiates insulin to up-regulate PROK1 in these cells. However, the androgens alone do not influence the expression of PROK1. Our findings suggest that insulin and androgens both are involved in the regulation of PROK1 that could have implications for normal and pathological pregnancies.     

Clonogenicity of human endometrial epithelial and stromal cells

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.     

Simvastatin decreases invasiveness of human endometrial stromal cells

Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.     

Phospholipase D1 as a key enzyme for decidualization in human endometrial stromal cells

Using primary cell cultures of human endometrial stromal cells (ES cells), we investigated the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which involves morphological and biological differentiation processes. When treated with 0.5 mM 8-Br-cAMP for 12 days, ES cells were transformed into a decidualized morphology and produced significant amounts of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Simultaneously, the activity and expression levels of PLD1 increased. In addition, removal of 8-Br-cAMP from decidualized ES cells restored the undifferentiated state, and this was accompanied by decreases in PLD1 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas PLD1 overexpression induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, knockdown of PLD1 by siRNA and blockage of PLD by treatment with 0.3% 1-butanol decreased PRL/IGFBP1 mRNA expression, whereas PLD1 overexpression increased PRL/IGFBP1 mRNA expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA expression and morphological changes, which implies that PA is an end-product of PLD activation-induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased PRL/IGFBP1 expression and inhibited morphological change, whereas pretreatment with propranolol caused no changes, as compared to cAMP-treated cells, which suggests that PA induces decidualization through phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br-cAMP-induced decidualization through PLA2G1B, and that PLD1 upregulation is essential for the decidualization of ES cells.     

In vitro decidualization of human endometrial stromal cells.

Stromal cells isolated from proliferative human endometrium undergo morphologic and biochemical changes when exposed to a mixture of ovarian hormones, acquiring characteristics of decidual cells. In addition to the previously reported progestin-induced secretion of prolactin (PRL) by explants of human proliferative endometrium, and of PRL and laminin by stromal cells in culture, "in vitro" induction of several other decidual cell products was demonstrated in the present study, using cultures of stromal cells isolated from proliferative endometrium. Incubation of stromal cells with a mixture of estradiol, medroxyprogesterone acetate and relaxin, at a concentration reported to yield maximal stimulation of PRL production, resulted in changes from elongated to rounder cells, approx. 90% of which showed immunostaining for PRL under these conditions. Immunocytochemical procedures were carried out on cytospins of decidual cells isolated from decidual tissue adherent to fetal membranes collected at delivery (positive controls), and on stromal cells cultured in Lab-Tek chamber-slides, in the absence (negative controls) or in the presence of added hormones. Antibodies to 24K (a heat-shock protein also named HRP27), desmin (present in intermediate filaments), p29 (a protein associated with the estrogen receptor), and PP12 (an insulin growth factor-1 binding protein), did not react with stromal cells isolated from proliferative endometrium but showed immunostaining of the rounder cells obtained after hormonal treatment when tested with the peroxidase-labeled second antibody complex. In another series of similar experiments, in which the same decidualization end-points were employed, changes in 24K, desmin and PP12 expression were obtained by adding to the insulin-containing medium PRL instead of the hormonal mixture, a finding suggesting sequential steps during the decidualization process.     

Prokineticin 1 is up‐regulated by insulin in decidualizing human endometrial stromal cells

Prokineticin 1 (PROK1), a hypoxia‐regulated angiogenic factor, has emerged as a crucial regulator of embryo implantation and placentation. Dysregulation of PROK1 has been linked to recurrent pregnancy loss, pre‐eclampsia, foetal growth restriction and preterm birth. These pregnancy complications are common in women with obesity and polycystic ovary syndrome, i.e. conditions associated with insulin resistance and compensatory hyperinsulinaemia. We investigated the effect of insulin on PROK1 expression during in vitro decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Insulin induced a significant dose‐dependent up‐regulation of PROK1 on both mRNA and protein level in decidualizing endometrial stromal cells. This up‐regulation was mediated by hypoxia‐inducible factor 1‐alpha (HIF1α) via the phosphatidylinositol 3‐kinase (PI3K) pathway. Furthermore, we demonstrated that PROK1 did not affect the viability, but significantly inhibited the migration of endometrial stromal cells and the migratory and invasive capacity of trophoblast cell lines. This in vitro study provides new insights into the regulation of PROK1 by insulin in human decidualizing endometrial stromal cells, the action of PROK1 on migration of endometrial stromal cells, as well as migration and invasion of trophoblasts. We speculate that hyperinsulinaemia may be involved in the mechanisms by which PROK1 is linked to placenta‐related pregnancy complications.     

Growth and hormonal responsiveness of human endometrial stromal cells in culture

The present review describes and discusses published results on growth and hormonal responsiveness of human endometrial stromal cells in culture. The proliferative potential of serially subcultured cells, that is, the number of cell doublings before cells enter mitotic senescence and cease to divide, was unusually high in stromal cells from several endometrial specimens, a property that may reflect the unique proliferative capacity of human endometrium when compared to other adult tissues. Fluorescent visualization of microfilaments revealed distinct age-related changes in the distribution of cytoskeletal fibers. Addition of ovarian steroids to the culture medium of stromal cells resulted in significant morphologic changes. From comparative studies using different culture media it became evident that medium components remarkably influenced cell morphology during early culture periods in an irreversible manner. Cultured stromal cells yielded interesting results in experiments designed to define the role of polyamines in growth regulation. Proliferation was greatly inhibited when polyamine levels were reduced by specific inhibition of ornithine decarboxylase, the first and rate limiting enzyme in polyamine synthesis which produces putrescine by catalytic conversion from ornithine. The antiproliferative effects were reversed by addition of putrescine to the culture medium. These results clearly establish a causal link between polyamine depletion and growth deficiencies and reveal an essential function of polyamines in stromal cell proliferation. Hormonally regulated parameters in cultured stromal cells include aromatase activity, pregnancy-associated plasma protein-A, 51K secreted protein, prolactin and laminin. The hormonally regulated production of prolactin and laminin, both considered markers of decidualization, together with morphologic changes of stromal cells to decidual-like cells, strongly suggest that human endometrial stromal cells, when subjected to appropriate hormonal stimulation, are capable of differentiating into decidual cells in culture. Cultured stromal cells therefore offer a unique opportunity to examine the complex changes in gene expression associated with decidualization. In addition, in vitro decidualization may prove to be an effective diagnostic tool in certain cases of infertility. Finally, decidualization of cultured stromal cells represents a relevant end point for testing compounds of potential clinical importance, such as synthetic progestins or antifertility drugs.     

In vitro hepatic differentiation of human endometrial stromal stem cells

Human endometrial stromal stem cells (hESSCs) can differentiate into mesodermal and ectodermal cellular lineages in the endometrium. However, whether hESSCs can differentiate into functional hepatic-like cells is unknown. In this study, we developed a multiple-step induction protocol to differentiate hESSCs into functional hepatic-like cells in vitro. Endometrial stromal cells were isolated by magnetic affinity sorting using anti-epithelial cell adhesion molecule-coated Dynabeads. The enriched hESSCs were analyzed by flow cytometry and were able to differentiate into osteoblasts or adipocytes under proper induction media. To differentiate into hepatic-like cells, hESSCs were cultured in a stepwise system containing hepatocyte growth factor, fibroblast growth factor-4, oncostatin M, and trichostatin A for a total of 24 d. The hepatic-like cell differentiation was analyzed by confocal microscopy and immunocytochemical staining. Glycogen storage, cellular urea synthesis, and ammonia concentrations were measured. Hepatic-like cells were successfully generated from hESSCs and were identified by their epithelial-like shape characteristics and expression of specific biomarkers albumin and cytokeratin 8 accompanied with a reduction of alpha-fetoprotein and alpha-smooth muscle actin expression. The hepatic-like cells generated were functional as evidenced by urea synthesis and glycogen storage. Our study demonstrated that hESSCs were able to differentiate into hepatic-like cells in vitro. Thus, endometrial stromal cells may be used as an easily accessible alternative source of stem cells for potential therapeutic applications in liver disease.     

Role of interleukin 1 in the regulation of cyclooxygenase gene expression in rat endometrial stromal cells.

Interleukin 1 alpha (IL-1 alpha) stimulates prostaglandin production and cyclooxygenase activity in endometrial stromal cells isolated from the uteri of ovariectomized rats that have been sensitized for the decidual cell reaction. The aim of the present study was to examine the effect of IL-1 alpha on the amount of cyclooxygenase mRNA and protein in these cells. Treatment with IL-1 alpha (20 ng ml-1) for 24 h significantly increased steady-state concentrations of cyclooxygenase 2 (COX-2) mRNA and protein in the cells, as determined by northern and western blot analyses, respectively. Cyclooxygenase 1 (COX-1) mRNA and protein were not detected. Dexamethasone (5 mumol l-1) prevented the IL-1 alpha-induced increase in COX-2 steady-state mRNA. Immunocytochemical staining of COX-2 in the treated cells indicated that IL-1 alpha increased staining, while dexamethasone inhibited this increase. Furthermore, the changes in staining were generalized and not confined to a small subpopulation of cells. These data demonstrate that IL-1 alpha increases steady-state concentrations of COX-2 mRNA and protein in endometrial stromal cells isolated from the uteri of rats that have been sensitized for decidualization.     

Progesterone metabolism in human endometrial stromal and gland cells in culture.

Metabolism of progesterone by human endometrium has been described, but the rapidity and extent of progesterone metabolism is incompletely documented in cellular fractions of normal endometrium. Therefore, we evaluated progesterone metabolism in separated stromal and gland cells in culture obtained from normal human endometrium by thin-layer chromatography. We find that in both cell types, the most abundant metabolite is 3beta-hydroxy-5alpha-pregnan-20-one (70%), followed by 5alpha-pregnane-3,20-dione (15%), and 3alpha-hydroxy-5alpha-pregnan-20-one (10%). A small amount is metabolized to 5alpha-pregnane-3alpha/3beta,20alpha-diols and to 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one. The metabolism of progesterone in cultured endometrial cells occurs rapidly; 70% of progesterone is metabolised in 8 h, and 90% by 24 h. We conclude that when in vitro experiments are conducted utilizing progesterone treatment, the rapidity and the extent of the metabolism of this steroid should be taken into account.     

Inhibition of cAMP-mediated decidualization in human endometrial stromal cells by IL-1beta and laminin.

Both IL-1 and laminin are reported to inhibit progesterone receptor-mediated decidualization signals in endometrial stromal cells, but the effects of these two inhibitors on cAMP-mediated decidualization signals remain unknown. Furthermore, the interaction between IL-1-stimulated and laminin-stimulated signals has not been analyzed yet. In this study, interactions between these two decidualization-inhibitory signals and their effects on 8-Br-cAMP-mediated decidualization in human endometrial stromal cells were investigated. Prolactin release from decidualized stromal cells was measured by enzyme immunoassay in order to quantitate in vitro decidualization. Both IL-1beta and laminin were found to inhibit 8-Br-cAMP-induced decidualization in vitro in human endometrial stromal cells. IL-1beta dose-dependently inhibited decidualization of stromal cells cultured on both laminin-coated and collagen-coated dishes, while laminin inhibited the decidualization of stromal cells cultured both with and without IL-1beta. These results indicate that IL-1beta and laminin additively and mutually inhibit cAMP-mediated decidualization signals, and that they may inhibit these signals through different mechanisms.     

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE₂ stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc.     

IGFBP-1 inhibits EGF mitogenic activity in cultured endometrial stromal cells

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.     

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.     

Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by estrogen and progesterone.

There is a cyclicity in the number of endometrial macrophages that is most likely secondary to changes in steroid hormone levels. One cytokine that controls macrophage migration is monocyte chemotactic protein-1 (MCP-1). In the endometrium, highest levels of MCP-1 are detected perimenstrually, when estrogen levels are low; however, when estrogen levels are high (around the time of ovulation), MCP-1 levels are lowest. We hypothesized that sex steroids may be involved in the regulation of macrophage migration by regulating MCP-1 expression. We investigated the regulation of MCP-1 expression in human endometrial stromal cells by estradiol 17beta (E2) and progestins. We found that MCP-1 mRNA levels decreased in response to E2 (5 x 10(-8) M), with biphasic nadirs at 8 h and 24 h. MCP-1 protein production was also inhibited by E2 in a concentration-dependent manner. Tamoxifen, an anti-estrogen, alone (10(-7) M) did not affect MCP-1 expression, but it reversed the E2-induced inhibition up to 80%. Progesterone (10(-7) M) alone slightly decreased MCP-1 levels, and the combination of E2 and progesterone further decreased them, but that decrease was not different from that observed using E2 treatment alone. In summary, we found that E2 inhibits MCP-1 expression in endometrial stromal cells, and we speculate that E2 may control endometrial macrophage migration by regulating MCP-1 expression.     

Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by integrin-dependent cell adhesion.

Shed menstrual endometrium is viable and has the ability to implant and grow in women, who eventually develop endometriosis. Many of the cell-to-cell or cell-to-extracellular matrix (ECM) connections are mediated by integrins. Monocyte chemotactic protein (MCP)-1, a potent chemotactic factor produced in many cell types, is elevated in the peritoneal fluid of women with endometriosis. In this study, we investigated whether endometrial stromal cell (ESC) adhesion itself induces the expression of MCP-1 and whether this process is integrin mediated. ESC were plated on Petri dishes and 24-well plates coated with fibronectin, laminin, collagen IV, poly-L-lysine, or mouse anti-human integrin beta(1) and beta(2) monoclonal antibodies. Adherence of ESC to various ECM substrates, except for poly-L-lysine, a non-integrin-dependent adhesion matrix, induced the expression of MCP-1 at both mRNA and protein levels. Engagement of beta(1)-containing integrins was associated with ESC adhesion and resulted in up-regulation of MCP-1 gene expression and protein secretion. Disruption of the actin cytoskeleton by treating ESC with cytochalasin D completely blocked the increase of MCP-1 induced in response to integrin activation. These findings indicate a novel mechanism of MCP-1 regulation. Cell adhesion to ECM is an important event that leads to stimulation of MCP-1 expression, and this process is mediated by integrins.