Loss of collagen type VI from rat endometrial stroma during decidualization.

The expression of collagen type VI in the extracellular matrix of rat uterine endometrial stroma after a decidual stimulus was examined by immunolocalization and immunoblotting. The intermediate filament protein, desmin, was used as a marker to identify decidual cells. Tissue was examined from pregnant animals and from ovariectomized, hormone-treated rats in which decidualization had been induced artificially. In undifferentiated tissue from both groups of animals, collagen type VI was abundant, and desmin was present only in vascular smooth muscle cells. By 72 h after a decidual stimulus, however, collagen type VI had essentially disappeared from the matrix of the antimesometrial stromal compartment, and desmin was highly expressed in the decidualizing cells. During regression of the decidual tissue, collagen type VI began to reappear in the stromal matrix, whereas desmin expression declined as decidual cells degenerated. These results indicate that remodeling of the uterine extracellular matrix in response to embryo implantation is a function of the differentiating decidual cell.     

Comparative analysis of subcellular fractionation methods for revealing α-actinin 1 and α-actinin 4 in A431 cells

α-Actinin 1 and α-actinin 4 are actin-binding proteins with shared structural functions that are responsible for the regulation of several processes in the cell. Based on previous data on the different distribution of these proteins in the nucleus and cytoplasm, we have studied in detail the presence of α-actinin 1 and α-actinin 4 in subcellular fractions in the A431 cells spread on fibronectin. The detection of α-actinins in some particular fractions has been shown to depend on the method of lysis, as well as whether the preliminary low-temperature freezing of cells was used. The application of various fractionation methods has allowed us to conclude that α-actinin 4 is present in all cytoplasmic and nuclear subfractions, whereas, in addition to in the cytoplasm, α-actinin 1 can also be revealed in the nuclear envelope fraction.     

Decidual cells produce a heparin-binding prolactin family cytokine with putative intrauterine regulatory actions

Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.     

Intermediate filament protein as a marker of uterine stromal cell decidualization

An increase in intermediate filaments has been reported in rat uterine stromal cells undergoing decidualization in vivo and in vitro. In order to identify biochemical correlates of this morphological change, we have identified (two dimensional gel electrophoresis, Western blots, indirect immunofluorescent staining) the constitutive intermediate filament proteins of stromal cells decidualizing in vivo and isolated stroma decidualizing in vitro as vimentin and desmin. Vimentin is common to all uterine stromal cells but increases, proportional to total cell protein, in decidualized stroma. Barely detectable in nondecidualized stroma, desmin, unlike vimentin, increases during decidualization at a rate greater than the increase in total cell protein. Neither the increase in vimentin or desmin is observed in hormonally sensitized, nondecidual stromal cells. Desmin, because it is selectively expressed in decidualizing stroma, could be considered unique enough to serve as a marker of decidual cell differentiation.     

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.     

Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo.

The effects of in vivo hormonal sensitization on the competence of uterine stromal (US) cells to decidualize in vitro were assessed. In vitro differentiation of uterine stroma isolated from Day 4 pregnant rats, sensitized to respond to a decidual stimulus, was compared to that in nonsensitized immature, castrated or cycling rats. The initiation of in vitro decidualization--as monitored by the expression of the decidual markers desmin and laminin in rat US cells--was independent of the hormonal status of the animal from which the cells were isolated and occurred in the absence of serum in the medium. Differentiation was accelerated in high-density cultures where contact inhibition suppressed proliferation and decreased the extent of cell growth. The extent to which in vitro decidualization imitates in vivo stromal cell differentiation was assessed by comparing decidualization in the rabbit, a species with only a limited decidual cell response, and in the rat. US cells isolated from nonpregnant rabbits differentiated in vitro by expressing laminin, but not desmin. Indirect immunofluorescence of frozen uterine sections from pregnant and nonpregnant rabbits validated in vitro differentiation as a faithful reflection of the in vivo program of decidualization. Although the program of US cell differentiation may vary between the species, initiation of differentiation in vitro appeared to be independent of hormonal preparation in vivo for both the species examined.     

Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy

We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.     

Alpha-actinin interactions with syndecan-4 are integral to fibroblast–matrix adhesion and regulate cytoskeletal architecture

All cells of the musculoskeletal system possess transmembrane syndecan proteoglycans, notably syndecan-4. In fibroblasts it regulates integrin-mediated adhesion to the extracellular matrix. Syndecan-4 null mice have a complex wound repair phenotype while their fibroblasts have reduced focal adhesions and matrix contraction abilities. Signalling through syndecan-4 core protein to the actin cytoskeleton involves protein kinase Cα and Rho family G proteins but also direct interactions with α-actinin. The contribution of the latter interaction to cell–matrix adhesion is not defined but investigated here since manipulation of Rho GTPase and its downstream targets could not restore a wild type microfilament organisation to syndecan-4 null cells. Microarray and protein analysis revealed no significant alterations in mRNA or protein levels for actin- or α-actinin associated proteins when wild type and syndecan-4 knockout fibroblasts were compared. The binding site for syndecan-4 cytoplasmic domain was identified as spectrin repeat 4 of α-actinin while further experiments confirmed the importance of this interaction in stabilising cell–matrix junctions. However, α-actinin is also present in adherens junctions, these organelles not being disrupted in the absence of syndecan-4. Indeed, co-culture of wild type and knockout cells led to adherens junction-associated stress fibre formation in cells lacking syndecan-4, supporting the hypothesis that the proteoglycan regulates cell–matrix adhesion and its associated microfilament bundles at a post-translational level. These data provide an additional dimension to syndecan function related to tension at the cell–matrix interface, wound healing and potentially fibrosis.     

Developmental expression of adenosine deaminase during decidualization in the rat uterus

Adenosine deaminase (ADA) is expressed in high concentrations at the fetal-maternal interface during postimplantation stages of gestation in the mouse. The experiments reported here were designed to identify the specific uterine cells that express ADA subsequent to implantation in the rat and to determine if embryonic cells contribute to ADA expression. The results of biochemical analysis demonstrate that ADA-specific activity increases to very high levels in implantation sites, beginning approximately 72 h after blastocyst attachment. Immunocytochemical analysis localized this ADA expression to the decidualized stromal cells in the antimesometrial region of the pregnant uterus. In experimentally induced deciduoma, these cells were capable of synthesizing high levels of both ADA and mRNA for ADA in the absence of embryos. The enzyme first appeared in decidual cell cytoplasm, approximately 72 h after induction of decidualization, and later was localized in the decidual cell nuclei. Since the expression of ADA and its mRNA in decidual cells follows the appearance of desmin, a protein marker for decidualization, by at least 48 h, ADA appears to be involved in the functioning of mature decidual cells rather than in stromal cell differentiation. The expression of ADA, but not desmin, was restricted to the antimesometrial decidual cells and decreased when these cells regressed. At mid-gestation ADA activity increased and was localized principally in the fetal placenta. The results presented here demonstrate that ADA is localized to the antimesometrial decidual cell and that its expression is consequent to differentiation of the uterine stromal cell and independent of any embryonic stimulus.     

腺病毒E4启动子结合蛋白-4(E4BP4)基因在小鼠胚胎着床期间子宫组织中的表达

腺病毒E4启动子结合蛋白-4(E4BP4)是哺乳动物细胞核内的一种碱性亮氨酸拉链(bZIP)型转录因子,参与调控细胞的存活和增殖。前期研究表明,它在孕第5天的小鼠着床位点有明显的高表达。本文分别应用Northem blot、in situ杂交、Western blot和免疫组织化学技术,对E4BP4基因在小鼠妊娠初始期子宫、着床期胚胎着床位点和非着床位点的表达情况进行了研究。观察发现：在小鼠妊娠初始期,E4BP4基因在子宫组织中的表达逐步上调;至胚胎着床期间,其在胚胎着床位点的表达水平进一步提高,并明显高于非着床位点;该基因的表达不依赖于胚胎,人工蜕膜化可诱导其表达：E4BP4 mRNA和E4BP4蛋白分子都主要分布于子宫腔周围的基质细胞和蜕膜细胞。上述结果提示E4BP4基因可能通过促进着床位点基质细胞的增殖和抑制蜕膜细胞的凋亡而参与胚胎着床过程的调控。     

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

Background  

During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-β isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.     

α-Actinin 1 and α-actinin 4: Contrasting roles in the survival, motility, and RhoA signaling of astrocytoma cells

α-Actinin is a prominent actin filament associated protein for which different isoforms exist. Here, we have examined whether the two highly homologous non-muscle α-actinin isoforms 1 and 4 exhibit functional differences in astrocytoma cells. The protein levels of these isoforms were differentially regulated during the development and progression of astrocytomas, as α-actinin 1 was higher in astrocytomas compared to normal brains whereas α-actinin 4 was elevated in high-grade astrocytomas compared to normal brains and low grade astrocytomas. RNAi demonstrated contrasted contributions of α-actinin 1 and 4 to the malignant behavior of U-373, U-87 and A172 astrocytoma cells. While α-actinin 1 appeared to favor the expansion of U-373, U-87 and A172 astrocytoma cell populations, α-actinin 4 played this role only for U-373 cells. On the other hand, downregulation of α-actinin 4, but not 1, reduced cell motility, adhesion, cortical actin, and RhoA levels. Finally, in the three astrocytoma cell lines examined, α-actinin 1 and 4 had contrasted biochemical properties as α-actinin 4 was significantly more abundant in the actin cytoskeleton than α-actinin 1. Collectively, these findings suggest that α-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells.     

Expression of desmin, laminin and fibronectin during in situ differentiation (decidualization) of rat uterine stromal cells

Immunocytochemical analysis of frozen rat uterine sections containing decidual tissue, formed in response to normal or artificial stimulation of uteri sensitized by endogenous or exogenous hormonal regimens, demonstrated an elevated expression of the intermediate filament protein desmin in decidual cells. Changes in the expression of extracellular matrix (ECM) components were coordinated with the elevated expression of desmin as stromal cells underwent decidualization. In parallel with the pattern of regional decidualization, as determined by elevated desmin expression, laminin accumulated in ECM of decidual cells while an apparent decrease in fibronectin was associated with altered organization at the decidual cell surface. The in situ observations confirm previous results, which indicated that the expression of desmin in decidual cells formed in vivo or in vitro is a valid marker of their differentiation, and resolve questions unanswered in the previous study: (a) desmin (and laminin) appear to be constitutively expressed in non-decidualized stroma at barely detectable levels, (b) desmin is a valid marker of stromal cell differentiation because it is expressed similarly in decidual cells, irrespective of varying experimental protocols for uterine sensitization and stimulation, and (c) desmin expression follows the same regional progression described for the process of decidualization in morphological and histochemical studies.     

Induction of cytokeratin expression in human mesenchymal cells

We studied the phenotypic features of some typical human mesenchymal cells, including decidual stromal cells and adult and fetal fibroblasts under different cell culture conditions by using antibodies to intermediate filament proteins and desmoplakins. In cell culture, the decidual stromal cells rapidly acquired typical fibroblastoid appearance with abundant arrays of vimentin filaments while the cytokeratin-positive epithelial cells, occasionally found in typical epithelioid colonies, lacked vimentin positivity and showed desmoplakin positivity. Within a few days, many of the stromal cells started to present cytokeratin positivity when cultured either in Condimed or in Chang medium. The cytokeratin positivity was first detected in small, scattered cytoplasmic dotted fibrils or in perinuclear dotlike aggregates with fibrillar projections. Later, denser cytokeratin-positive fibrillar arrays could also be seen in stromal cells, which lacked desmoplakin positivity as judged by two monoclonal antibodies. Decidual stromal cells were also cloned and in five out of ten clones some of the cells acquired a similar cytokeratin positivity when transferred into Chang or Condimed medium. Immunoblotting results indicated that cytokeratins 8, 18, and 19 can be found in these cultures. Similar cytokeratin positivity could also be seen in the same culture conditions in cultured fetal fibroblasts from skin, chorionic villi, and lung but not in young or adult skin fibroblast cultures. The present results suggest that decidual stromal cells as well as some embryonal mesenchymal cells can acquire epithelial differentiation in vitro as judged by the emergence of cytokeratin proteins. This ability appears to be lost in the corresponding adult cell. The results furthermore suggest that cytokeratin fibrils can be organized in the cytoplasm without an apparent organization center and that neither the appearance of desmoplakins nor the formation of cell-to-cell contacts are required for cytokeratin filament assembly.     

Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15

The natural killer (NK) cells that are present in the uterine mucosa (decidua) during early pregnancy have a distinctive phenotype, CD56(bright) CD16(-). These cells have previously been shown to proliferate and be activated by interleukin (IL)-2. However, IL-2 is absent from the decidua and placenta, and we have therefore investigated whether IL-15 is present in the uterus and can act on decidual NK cells. Both IL-15 mRNA and protein were found in a variety of cells but particularly in decidual macrophages. IL-15 induced a proliferative response in decidual NK cells that was blocked by anti-IL-15 and was augmented by stem cell factor. The cytolytic activity of decidual NK cells against K562 was augmented. Interestingly, in contrast to IL-2, although activation with IL-15 resulted in some killing of JEG-3 choriocarcinoma cells, normal trophoblast cells remained resistant to lysis. These findings suggest that IL-15 is a candidate cytokine responsible for NK cell proliferation in vivo in the progesterone-dominated secretory endometrium and early decidua.     

Mouse endometrial stromal cells produce basement-membrane components

During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The decidualization of stromal cells is characterized by the accumulation of pericellular basement-membrane material. Decidualized stromal cells of pregnancy differ in this respect from stromal cells obtained from nonpregnant uteri, which are not associated with any significant amounts of basement-membrane components. Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells synthesize laminin, entactin, fibronectin, type-IV collagen, and heparan sulfate proteoglycan. Stromal cells isolated from nonpregnant uteri and explanted in vitro produced the same basement-membrane components. Apparently, these cells were stimulated by the procedure used during isolation and explanation to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components.     

Direct observation of α-actinin tension and recruitment at focal adhesions during contact growth

Adherent cells interact with extracellular matrix via cell–substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion?s growth.     

Evidence for a role for a uterine renin-angiotensin system in decidualization in rats.

Experiments were performed in vivo and in vitro to determine the effects of enalaprilat, a specific inhibitor of angiotensin-converting enzyme, on various aspects of the decidual cell reaction in rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For in vivo experiments, intrauterine infusions of enalaprilat alone, and in combination with angiotensin II and prostaglandin E2 (PGE2), were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations, endometrial vascular permeability, alkaline phosphatase activity and uterine weight that occurred sequentially following infusion of vehicle. Concurrent infusion of angiotensin II did not reverse any of these inhibitory effects; PGE2 infusion partially, but not completely, reversed the inhibition of increase in uterine weight, although it did not alter the inhibition of endometrial vascular permeability. For in vitro experiments, endometrial stromal cells were obtained from uteri on the day of sensitivity and cultured for up to 3 days in the presence of enalaprilat and angiotensin II. Enalaprilat inhibited in a dose-dependent manner the increases in stromal cell alkaline phosphatase activity and media PGE concentration that occurred in the control cultures; these effects were fully reversed by concurrent treatment with angiotensin II. The inhibition of stromal alkaline phosphatase activity was also reversed by PGE2; conversely, the ability of angiotensin II to reverse the effect of enalaprilat was lost in the presence of indomethacin. These studies provide evidence of a requirement for angiotensin II during the decidual cell reaction in rats and suggest that it acts, at least in part, through a PG-dependent mechanism.