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Biomedical subjects

B Babiarz

Publications and source records attributed to B Babiarz.

11 recordsLinked to original sources
PubMed1999

Expression of cathepsin proteinases by mouse trophoblast in vivo and in vitro.

Implantation and placentation in the mouse requires successful invasion of the uterine wall by primary and secondary trophoblast giant cells. Their invasive nature depends in part on the upregulation of proteinases for the phagocytosis and extracellular digestion of maternal cells and matrix materials. The work reported here studies the expression of cathepsin proteinases during secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. Cathepsins B (CB), L (CL), and D (CD) were found to be upregulated during trophoblast differentiation in vivo at the message and protein level producing expression patterns equivalent to those of primary trophoblast. Invasive trophoblast cells expressed higher levels of the processed or active forms of the enzymes, coinciding with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials. Trophoblast differentiation in vitro showed a similar upregulation of cathepsin enzymes. The enzymes were localized to heterogeneous vesicles that resembled both lysosomes and heterophagic vesicles. The presence of a large lysosomal population within the giant cells was confirmed by vital staining with acridine orange. Analysis of trophoblast-conditioned media also demonstrated secreted forms of CB and CL. The results suggest that cathepsin enzymes may contribute to trophoblast invasion not only through the intracellular breakdown of molecules phagocytosed by trophoblast cells, but also by the extracellular digestion of matrix molecules and activation of other pro-enzymes.

Animals↗
PubMed1997

The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation.

The implantation of the mouse embryo requires the controlled invasion of the uterine stroma by the embryonic trophoblast. This event is dependent, in part, on the secretion of matrix metalloproteinases and serine proteinases for the extracellular degradation of the uterine matrix. Proteinase activity is controlled by stromal decidualization and specific proteinase inhibitors. This work adds to our understanding of implantation and placentation by reporting the expression and function of another class of proteinases/inhibitors closely related to invasive cell behavior. We focused on the cysteine proteinases, cathepsins B and L, and their inhibitor cystatin C. Northern blots showed that trophoblast expressed cathepsin B throughout the invasive period (days 5.5-10.5). Both cathepsin B message and cathepsin L protein were localized to the mature, invasive trophoblast giant cells. Substrate gel electrophoresis showed an increase in giant cell cathepsin activity with enzyme profiles changing at the end of the invasive period. Northern and western blotting showed that cystatin C, the main inhibitor of cathepsins, was a major product of the decidualizing stroma. Message levels first increased in peripheral decidualizing cells, with the protein localizing close to the embryo during implantation (days 5.5-8.5). With the regression of the decidua beginning on day 9.5, a coordinated upregulation of both cathepsin B and cystatin C was observed implying a role for controlled cathepsin expression during apoptosis. E-64, a synthetic inhibitor of cathepsins B and L, was injected into pregnant females at the stage of blastocyst attachment (days 4.5-5.5). High doses resulted in the complete failure of implantation while lower doses resulted in stunted embryos and a reduced decidual reaction. These results suggested that cathepsins B and L are necessary for normal embryo development and uterine decidualization, and that decidua contributes to their control by a coordinated expression of cystatin C within the implantation site.

Animals↗
PubMed1996

Localization and expression of fibronectin during mouse decidualization in vitro: mechanisms of cell:matrix interactions.

During implantation, the embryonic trophoblast aggressively invades the uterine stroma. The resulting uterine reaction, decidualization, involves differentiation of new cell morphologies and remodeling of the extracellular matrix. This creates an environment that first permits invasion, then controls this invasion to allow the establishment of the placenta. The production, organization, and cellular interactions with the matrix are thought to underlie decidual functions. We have begun a reductional analysis of the components of the decidual matrix, focusing on extracellular fibronectin (FN). Using decidual cell cultures prepared from day 7 implantation sites, the synthesis, extracellular organization, and details of decidual cell:FN interaction were studied. Employing immunofluorescence, immunoprecipitation, and dot blot analysis, decidualizing cultures showed a constitutive level of FN synthesis and deposition. The differentiating cells organized extracellular FN in patterns similar to that seen in vivo. The predominant, flattened dendritic decidual cells organized FN in long, thin fibrils. Large, rounded decidual cells, limited to the primary decidual zone in vivo, showed FN limited to punctate membrane patches and short, thick fibrils. Using double labeling techniques, FN expression was co-localized with actin microfilament (MF) bundles during the cytoskeletal changes associated with the differentiation of both decidual cell types. The function of MFs in maintaining morphology was demonstrated by cytochalasin B perturbation. Attachment of decidual cells to FN was calcium dependent and gly-arg-gly-asp-ser-pro (GRGDSP) sensitive, with dendritic decidual cells expressing the alpha 5 and beta 1 integrin subunits. This suggests that an integrin system functions to attach decidual MF bundles to extracellular FN. This work shows that during decidual matrix remodeling, constitutive levels of FN are maintained to provide an extracellular framework to stabilize the decidual cytoskeleton and support morphological differentiation of decidual cells.

Animals↗
PubMed1996

An in vitro system for the study of matrix metalloproteases during decidualization in the mouse.

Decidualization results in the remodeling of the extracellular matrix with the loss of collagen type I and the appearance of basement membrane matrix components. We have developed an in vitro assay system to study matrix metalloproteases during mouse decidualization. Uterine stroma, or decidua isolated from day 7.5 pregnant mice, were grown on a three-dimensional collagen type I matrix (Vitrogen). Gelatin zymography of conditioned media from these cultures showed constitutive secretion of processed forms of gelatinase A at 65, 62, and 59 kDa with 62 kDa predominating. Similar patterns of gelatinase A expression were obtained from tissue lysates of decidualizing uteri from days 5.5 to 7.5 of development. Cells cultured on Vitrogen, but not on plastic or matrix-coated dishes, were able to process the proenzyme to the 59 kDa form as observed in vivo. Only stroma cells cultured on a coating of collagen type I displayed the same increase in the 59 kDa zymogen. Decidua cells grown on Vitrogen attached and then migrated into aggregates that eventually penetrated the gel and spread as differentiated decidua on the underlying plastic. These preliminary results suggested that the in vitro assay system can be used to study the role of metalloproteases in matrix remodeling during decidualization.

Animals↗
PubMed1993

Mechanisms of murine trophoblast interaction with laminin.

Implantation of the mouse embryo depends on the interaction of the trophoblast and the uterine environment, which includes an extracellular matrix with abundant amounts of laminin. The experiments reported here analyzed the interaction of trophoblast with laminin in vitro. Secondary trophoblast attachment and spreading on laminin matrices was competitively inhibited in the presence of the laminin peptides YIGSR (Tyr-Ile-Gly-Ser-Arg), RGD (Arg-Gly-Asp), and IKVAV (Ile-Lys-Val-Ala-Val). Substrates of multiple, but not single, peptides supported attachment and spreading of trophoblast. The presence of laminin-binding proteins on trophoblast cell surfaces were detected by immunofluorescence using antiserum directed against a fusion protein encoded by the full-length clone for the 32-kDa laminin-binding protein. Immunoblotting with the same antiserum identified immunoreactive proteins of 33, 41, 45, 62, and 76 kDa in trophoblast lysates. The antiserum also inhibited the spreading of trophoblast on laminin matrices.

Amino Acid Sequence↗
PubMed1992

3T3 cell surface galactosyltransferase is a calcium-dependent adhesion molecule for collagen type IV.

Cell surface galactosyltransferase (GalTase) has been previously shown to mediate cell spreading or migration on laminin matrices. This work demonstrates that 3T3 cell surface GalTase also mediates cell attachment to collagen type IV. Attachment to collagen type IV was blocked by perturbations of GalTase or substrate pregalactosylation on cells possessing only calcium-dependent mechanisms of adhesion. Cells with both calcium-dependent and calcium-independent systems were not affected by GalTase perturbation. Collagen type IV was shown to possess GalTase substrates since matrices could be galactosylated by both soluble enzyme and 3T3 cells.

3T3 Cells↗
PubMed1990

The role of murine cell surface galactosyltransferase in trophoblast: laminin interactions in vitro.

Implantation of the mouse embryo involves the invasion of the secondary trophoblast giant cells of the ectoplacental cone (EPC) into the uterine decidua. The mechanisms of this event are poorly understood. The putative substrate molecules found in the decidua which could support trophoblast invasion include laminin, fibronectin, and collagen type IV. EPCs dissected from Day 7.5 embryos were cultured on all three matrices. Galactosyltransferase (GalTase) was localized by immunolabeling on trophoblast cell surfaces when grown on laminin but not the other matrices. Perturbations of the enzyme:substrate complex with alpha-lactalbumin, uridine diphosphogalactose, anti-GalTase, and pregalactosylation of the matrix did not affect rates of EPC attachment. However, decreased rates of migration or altered morphologies of spreading cells were observed. Laminin, and not fibronectin or collagen type IV, could be galactosylated with both exogenous GalTase or EPC outgrowths. Digests of galactosylated laminin produced a glycoconjugate substrate with a molecular weight of less than 10K. The results suggest that invasive secondary trophoblast cells possess a GalTase-mediated migration system that is functional on laminin.

Animals↗
PubMed1984

Expression of a glucose-regulated cell surface protein in early mouse embryos.

A 100,000-Da glucose-regulated surface protein (100K-GRP) has previously been isolated from the cell surface and culture medium of human fibroblasts. A rabbit antiserum directed against this protein reacts with the cell surface of both human and murine cultured cells and with a broad spectrum of mammalian tissues. It is shown, via indirect immunofluorescence, that this protein is also present on cells of the developing mouse embryo and can be detected as early as the 4-cell stage. The 8-cell embryo and morula show positive surface labeling; the inner cell masses of both the pre- and postimplantation blastocysts are also positive but the trophectoderm is not. At the 6-day egg cylinder stage, the embryonic and extra-embryonic ectoderm label intensely with the antiserum and visceral endoderm shows faint labeling. No labeling can be detected on parietal endoderm or on the trophoblastic giant cells invading the uterine decidua. However, the internal cells of the ectoplacental cone exhibit bright fluorescence. The same pattern is observed on 7- to 8.5-day embryos, except that at this stage no label is associated with the visceral endoderm. In addition, mesodermal cells emerging from the primitive streak are also labeled.

Animals↗