Putative role for EPC-1/PEDF in the G0 growth arrest of human diploid fibroblasts

EPC-1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast-like (HDF) cells and is diminished with proliferative senescence in vitro. EPC-1 expression in HDF cells is induced under conditions of density-dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC-1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC-1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC-1 antibodies alone enhance the ability of near plateau-phase early PDL WI-38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC-1 (rEPC-1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC-1 expression in WI-38 and IMR-90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC-1 expression with SV40 transformation occurs at the level of steady-state mRNA and protein accumulation with genomic EPC-1 sequences grossly intact. Taken together, these results suggest that EPC-1 may play a role in the entry of early passage fibroblasts into a G(0) state or the maintenance of such a state once reached.     

The role of interleukin-1 in interactive senescence and age-related human endometrial cancer

The causes of the age-related increase in cancer rates are poorly understood. One cause could be age-related changes in the stromal/epithelial cell interactions that facilitate tumorigenesis. We tested the hypothesis that aging of human endometrial stromal fibroblasts (ESF) alters their influence over endometrial epithelial cells. ESF from adults were found to inhibit anchorage-independent proliferation, to restrain colony outgrowth, and to induce formation of normal tissue architecture by human endometrial cancer cells. As ESF age, these inhibitory influences on malignant-like behaviors by epithelial cells are altered, becoming stimulatory. Age-related change in interleukin-1alpha (IL-1alpha) expression is a molecular determinant of ESF/epithelial cell interactions. Levels of IL-1alpha and IL-1-induced mRNAs increase in ESF with age. Treatment with IL-1 accelerates age-related changes in mRNA abundance and loss of ESF restraint over malignancy-associated behaviors by epithelial cells. Transfection of ESF with the intracellular IL-1 receptor antagonist preserved the young phenotype with respect to interactions with epithelial cells and prevented age-associated increases in groalpha and IL-8 mRNA levels. Our results indicate that aging of ESF is accompanied by an interactive senescence that alters ESF signaling to cancer cells and could contribute to increased cancer rates by providing a microenvironment that is more conducive to tumorigenesis.     

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in G1. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (M1) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed M1 mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active M1 mechanism.     

C-ras expression decreases during in vitro senescence in human fibroblasts

Expression of the c-ras oncogene was determined in growing early and late passage human (IMR-90) fibroblasts using northern blot analysis of total cellular RNA. It was found that late passage cells demonstrated lower levels of c-ras by about four fold when compared to levels found in early passage cells. Southern blot analysis of genomic DNA from early and late passage fibroblasts digested with either SacI or BamHI showed somewhat increased hybridization levels in early passage cells compared to late passage cells. Data is discussed in relation to a previous report of c-ras expression and gene amplification.     

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.     

Superoxide dismutase 1 knock-down induces senescence in human fibroblasts

Reactive oxygen species (ROS) such as superoxide radicals are responsible for the pathogenesis of various human diseases. ROS are generated during normal metabolic process in all of the oxygen-utilizing organisms. The copper-zinc-containing SOD (SOD1) acts as a major defense against ROS by detoxifying the superoxide anion. In model organisms, SOD1 has been shown to play a role in the aging process. However, the exact role of the SOD1 protein in the human aging process remains to be resolved. We show that SOD1 RNA interference (RNAi) induces senescence in normal human fibroblasts. This premature senescence depends on p53 induction. In contrast, in human fibroblastic cells with inactivated p53, the SOD1 RNAi is without effect. Surprisingly, in cancer cells (HeLa), the SOD1 RNAi induces cell death rather then senescence. Together, these findings support the notion that in normal human cells the SOD1 protein may play a role in the regulation of cellular lifespan by p53 and may also regulate the death signals in cancer cells.     

Suppression of thioredoxin-1 induces premature senescence in normal human fibroblasts

Thioredoxin (TRX) is a ubiquitous multifunctional thiol protein that is critically involved in maintaining cellular redox homeostasis. Levels of thioredoxin-1 (TRX1), the major isoform of TRX, have been shown to correlate with organismal lifespan and age-associated tissue deterioration. Accordingly, we investigated the direct functional effects of suppressing TRX1 levels on cellular senescence, a phenomenon intimately linked with tissue degeneration and aging. Here we find that suppression of TRX1 expression via shRNA rapidly induces premature senescence in young human skin fibroblasts through upregulation of the p53/p21^Cip1/Waf1 and p16^INK4a tumor suppressor pathways. Moreover, inhibition of these pathways by introduction of SV40 Large T Antigen prevents TRX1 suppression-induced premature senescence but not susceptibility to oxidative stressors. Thus our results suggest that TRX1 has a role in suppressing senescence in normal cells in addition to its function as a redox-protective protein.     

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.     

Stress-induced premature senescence in BJ and hTERT-BJ1 human foreskin fibroblasts

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric DNA-binding complex of the subunits alpha and beta with relevance in O(2) and energy homeostasis. The labile component, HIF-1alpha, is not only activated by hypoxia but also by peptides such as insulin and interleukin-1 (IL-1) in normoxia. We investigated whether inhibitors of mitogen-activated protein kinase kinases (MAPKKs: PD 98059, U0126) and phosphatidylinositol 3-kinase (PI3K: LY 294002) do not only lower the hypoxia-induced, but also the insulin- and IL-1-induced HIF-1alpha accumulation and HIF-1 DNA-binding in human hepatoma cell cultures (line HepG2). The results show that LY 294002 suppressed HIF-1 activation in a dose-dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF-1, the production of erythropoietin was fully blocked and that of vascular endothelial growth factor reduced following inhibition of the PI3K pathway. The role of MAPKKs in this process remained in question, because PD 98059 and U0126 did not significantly reduce HIF-1alpha levels at non-toxic doses. We propose that PI3K signaling is not only important in the hypoxic induction of HIF-1 but it is also crucially involved in the response to insulin and IL-1.     

Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts

Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.     

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.     

Extracellular matrix-dependent regulation of Fas ligand expression in human endometrial stromal cells.

Interaction between endometrial stromal cells and extracellular matrix (ECM) components has a crucial role in the development of endometriosis. Endometrial stromal cells attach to the mesothelial surface of peritoneum by means of integrins during their initial implantation and growth in endometriosis. Similarly, interaction between integrin and the extracellular matrix is also crucial for the remodeling of the endometrium during early pregnancy. We hypothesized that adhesion of endometrial stromal cells to the extracellular matrix could suppress the immunologic reaction to implanting endometrial cells by inducing the expression of Fas ligand (FasL), a mediator of the apoptotic pathway. Western blot analysis of human endometrial stromal cells plated onto fibronectin, laminin, and collagen IV revealed higher levels of FasL protein expression compared with endometrial stromal cells that plated to BSA-coated plates (control). Immunocytochemistry results from endometrial stromal cells plated to extracellular matrix proteins demonstrated a similar up-regulation of FasL expression. Eutopic endometrial stromal cells from women with endometriosis demonstrated higher FasL expression on control plates and those coated with extracellular matrix proteins compared with those from women without endometriosis. Disruption of actin cytoskeleton in endometrial stromal cells by treatment with cytochalasin D blocked the increase of FasL protein expression that occurred in response to adhesion to the extracellular matrix. These results suggest that attachment of endometrial stromal cells during retrograde menstruation to a new environment such as peritoneum with increased expression of laminin, fibronectin, and collagen IV could lead to an increase in FasL expression. Induction of FasL expression by adhesion of endometrial stromal cells to the extracellular matrix may take part in the development of a relative immunotolerance by inducing apoptosis of cytotoxic T lymphocytes, which will allow further development of ectopic implants.     

Tibolone exerts progestational inhibition of matrix metalloproteinase expression in human endometrial stromal cells

Tibolone and its metabolites were evaluated on matrix metalloproteinase (MMP) expression in human endometrial stromal cells (HESCs) under the hypothesis that these steroids would act as progestins on MMP-1, -2, and -3 expression. After 7 days of priming and 24h experimental incubation of confluent cultured HESCs, 10(-7) M medroxyprogesterone acetate (P) reduced MMP-1 to 49+/-34% (p<0.05) and MMP-3 to 33+/-22% of basal levels (mean+/-S.E.M., p<0.05, n=5). Although HESCs were unaffected by 10(-8) M estradiol (E), E+P reduced MMP-1 and MMP-3 levels an additional 2.5-fold from P alone. Tibolone and Delta-4 tibolone were equivalent to E+P in inhibiting MMP-1 and MMP-3 output, whereas 10(-6)M of 3alpha-OH or 3beta-OH tibolone was required to elicit significant inhibition of both MMPs (p<0.05). By contrast, none of the treatments affected HESC-secreted MMP-2 output. The ELISA results were confirmed by Western blotting and by substrate gel zymography. Quantitative RT-PCR demonstrated corresponding changes in MMP-1 and MMP-3 mRNA levels. Inhibition of MMP-1 and MMP-3 expression by tibolone and Delta-4 tibolone is consistent with the metabolism of tibolone to Delta-4 tibolone, and subsequent binding of Delta-4 tibolone to the progesterone receptor. Since 3alpha-OH and 3beta-OH tibolone bind exclusively to the estrogen receptor, their inhibition of MMP-1 and MMP-3 suggests metabolism by HESCs to Delta-4 tibolone. These observations help to explain the paradox that the endometrium becomes atrophic after tibolone administration despite the persistence in the circulation of 3alpha-OH and 3beta-OH tibolone, but not tibolone or Delta-4 tibolone.     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.