Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep

The extracellular matrix protein osteopontin (OPN) is a component of histotroph that increases in uterine flushings from pregnant ewes during the peri-implantation period and is localized on the apical surfaces of the uterine luminal epithelium (LE) and conceptus trophectoderm (Tr). The potential involvement of OPN in the implantation adhesion cascade in sheep was investigated by examining temporal, spatial, and potential functional relationships between OPN, Muc-1, and integrin subunits during the estrous cycle and early pregnancy. Immunoreactive Muc-1 was highly expressed at the apical surfaces of uterine luminal (LE) and glandular epithelium (GE) in both cycling and pregnant ewes but was decreased dramatically on LE by Day 9 and was nearly undetectable by Day 17 of pregnancy when intimate contact between LE and Tr begins. In contrast, integrin subunits alpha(v), alpha(4), alpha(5), beta(1), beta(3), and beta(5) were constitutively expressed on conceptus Tr and at the apical surface of uterine LE and GE in both cyclic and early pregnant ewes. The apical expression of these subunits could contribute to the apical assembly of several OPN receptors including the alpha(v)beta(3), alpha(v)beta(1), alpha(v)beta(5), alpha(4)beta(1), and alpha(5)beta(1) heterodimers on endometrial LE and GE, and conceptus Tr in sheep. Functional analysis of potential OPN interactions with conceptus and endometrial integrins was performed on LE and Tr cells in vitro using beads coated with OPN, poly-L-lysine, or recombinant OPN in which the Arg-Gly-Asp sequence was replaced with RGE or RAD. Transmembrane accumulation of talin or alpha-actinin at the apical surface of uterine LE and conceptus Tr cells in contact with OPN-coated beads revealed functional integrin activation and cytoskeletal reorganization in response to OPN binding. These results provide a physiological framework for the role of OPN, a potential mediator of implantation in sheep, as a bridge between integrin heterodimers expressed by Tr and uterine LE responsible for adhesion for initial conceptus attachment.     

Epithelial-stromal tissue interaction in paramesonephric (Müllerian) epithelial differentiation.

During organogenesis, the middle to caudal portion of Müllerian epithelium differentiates into uterine and vaginal epithelia in females. Functional differentiation of uterine and vaginal epithelia occurs in adulthood, and is regulated by 17beta-estradiol (E(2)) and progesterone. In this report, the roles of mesenchyme/stroma in differentiation of uterine and vaginal epithelia were studied in tissue recombination experiments. At birth, Müllerian epithelium was negative for uterine and vaginal epithelial markers. Tissue recombinant experiments showed that uterine and vaginal gene expression patterns were induced in neonatal Müllerian epithelium by the respective mesenchymes. Differentiated adult uterine and vaginal epithelia did not change their original gene expression in response to heterotypic mesenchymal induction. In the adult vagina, E(2) induced expression of involucrin, a CCAAT/enhancer-binding protein beta and cytokeratin 1 via estrogen receptor alpha (ERalpha). Tissue recombination experiments with wild-type and ERalpha knockout mice demonstrated that epithelial gene expression is regulated by E(2) via epithelial-stromal tissue interactions. Uterine/vaginal heterotypic tissue recombinations demonstrated that functional differentiation of uterine and vaginal epithelia required organ-specific stromal factors. In contrast, stromal signals regulating epithelial proliferation appeared to be nonspecific in the uterus and vagina.     

Organogenesis of the exocrine gland

Morphogenesis of exocrine glands is a complex stepwise process of epithelial ingrowth, ductal elongation, ductal branching, and alveolar or acinar differentiation. Emerging from an increasing number of mouse gene knockout, dominant-negative, and antisense models is the identification of a remarkable collection of cell adhesion molecules, growth factors, and their receptors whose time-dependent contributions to glandular organogenesis are essential. Many have cryptically overlapping and interdependent but noncompensatory roles. Discoidin domain receptor 1 tyrosine kinase (DDR1) and the ErbB1 receptor of amphiregulin are, for example, required for ductal branching and elongation. Each is in turn dependent on the Wnt family of morphogenic factors for autophosphorylation or transactivation, respectively. Here we review the current cast of exocrine glandular morphogens, as a foundation for a global or systems biology appreciation of the interweaving signaling pathways that underlie mammalian glandular morphogenesis.     

Tyrosine kinase inhibition decreases Muc-1 expression in mouse epithelial cells

Mouse uterine epithelial cells (UEC) express high levels of both messenger RNA (mRNA) and protein encoding the polymorphic mucin glycoprotein, Muc-1, under most conditions in vivo and in vitro. Although steroid hormones modulate Muc-1 expression in vivo, it is not clear if these actions are mediated directly by steroid hormone receptors or indirectly by modulation of key intracellular signal transduction cascades. To address the latter issue, we examined the effects of a wide variety of modulators of signal transduction cascades on the expression of Muc-1 in primary cultures of polarized mouse UEC. Transient exposure of UEC to agents that inhibit tyrosine kinases by distinct mechanisms, i.e., tyrphostin, genistein, and staurosporine, consistently and significantly reduced Muc-1 expression. In contrast, a variety of agents that modulate protein kinase A- or C-dependent pathways had little or no effect on Muc-1. The effect of tyrphostin proved to be similar in magnitude at both the level of Muc-1 protein and mRNA expression. Transient transfection assays of mouse UEC and a murine mammary epithelial cell line, NMuMG, with mouse Muc-1 promoter-CAT reporter constructs demonstrated a similar (50–60%) degree of tyrphostin inhibition. These observations suggested an action at the level of Muc-1 gene expression. Levels of 100,000g soluble tyrosine kinase activity in mouse UEC freshly isolated from estrous stage (high-level Muc-1 expression) and day 4 of pregnancy (low-level Muc-1 expression) correlated with Muc-1 expression. Furthermore, pretreatment of day 4 pregnant mice with the anti-progestin, RU486, an agent previously shown to restore or maintain high levels of Muc-1 expression, also restored soluble tyrosine kinase activity to levels similar to that observed in estrous stage mice. Collectively, these results indicate that tyrosine kinase activity is required to maintain high level Muc-1 expression in mice. J. Cell. Physiol. 170:200–208, 1997. © 1997 Wiley-Liss, Inc.     

Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism.

We present here the full-length cDNA sequence and genomic structure of the mouse homologue of the tumor-associated mucin, MUC1. This mucin (previously called polymorphic epithelial mucin) is present at the apical surface of most glandular epithelial cells. The mouse gene, Muc-1, encodes an integral membrane protein with 40% of its coding capacity made up of serine, threonine, and proline, a composition typical of a highly O-glycosylated protein. The mucin core protein consists of an amino-terminal signal sequence, a tandem repeat domain encoding 16 repeats of 20-21 amino acids, and unique sequence containing transmembrane and cytoplasmic domains. Homology with the human protein is only 34% in the tandem repeat domain, mainly showing conservation of serines and threonines, presumed sites of O-linked carbohydrate attachment. Homology rises to 87% in the transmembrane and cytoplasmic domains, suggesting that these regions may be functionally important. The pattern of expression of the mouse mucin is very similar to that of its human counterpart and accordingly the two promoter regions share high homology, 74%, although previously identified potential hormone-responsive elements are not conserved. Interestingly, the mouse homologue, unlike its human counterpart does not exhibit a variable number tandem repeat polymorphism. We present evidence that suggests that the mouse gene was at one time polymorphic but has mutated away from this state.     

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate

 Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5–15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell–cell communication, cell fate, and differentiation of conducting airway epithelia. Accepted: 5 May 1997     

Expression of p53 in luminal and glandular epithelium during the growth and regression of rat uterus during the estrous cycle

It has been well recognized that epithelial cells of the rat endometrium cyclically proliferate and die during the estrous cycle. The aim of the present study was to determine p53 expression pattern and correlate it with the the apoptotic pattern of epithelial cells of the rat uterus during the estrous cycle. The p53 mRNA and protein expression pattern was assessed by in situ hybridization and immunohistochemistry. The apoptotic index was determined by using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and electron microscopy. The highest p53 mRNA content, detected by in situ hybridization, was observed on the metestrus day both in the luminal and the glandular epithelia. During this period both epithelia presented high proliferation. The content of p53 mRNA markedly decreased in the following days, presenting its minimal values on the estrus day. The highest number of p53 immunopositive nuclei, in both the luminal and the glandular epithelia, was also detected on the metestrus day, while the lowest one was found on estrus day. On the proestrus day, p53 protein was predominantly detected in the glandular epithelium. However, on the estrus day, p53 protein was detected both in the nuclei and in the cytoplasm of luminal epithelial cells, predominantly in the cytoplasm. The highest apoptotic index in both the luminal and the glandular epithelia was observed on the estrus day whereas the lowest one was observed on the proestrus day. The apoptotic index values were higher in the luminal than in the glandular epithelia. The overall results indicate that p53 expression at both mRNA and protein levels is higher on the metestrus day when the apoptotic index is low. This suggests that p53 should play an important physiological role during proliferative phases of the estrous cycle in the rat uterus.     

Stage-specific expression of the intermediate filament protein cytokeratin 13 in luminal epithelial cells of secretory phase human endometrium and peri-implantation stage rabbit endometrium

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.     

Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland epithelium

The expression of the apoptosis-related proteins Bcl-2 and Bax was investigated by immunohistochemistry in the normal non-lactating human mammary gland in relation to cell proliferation and apoptosis. In order to characterize individual Bax/Bcl-2-immunoreactive cells, the epithelial markers cytokeratin 14 and 19 and the macrophage marker CD 68 were used. Secretory-like differentiation of epithelial cells was characterized by histochemistry and lectin staining of surface glycoconjugates. Cell proliferation was exclusively found in glandular epithelial cells with broad contact to the ductular lumen, whereas nuclei with apoptosis-related DNA fragmentation were seen predominantly in basally located glandular epithelial cells and in myoepithelial cells. Weak immunoreactivity for Bcl-2 and Bax was present throughout all epithelia, suggesting a balance between pro- and antiapoptotic effects in the majority of epithelial cells. However, specific cells showed a strong staining for Bax or Bcl-2. The strongly Bcl-2-immunoreactive epithelial cells were not identical with proliferating cells, but they resembled them in configuration and in the luminal intraepithelial position. In contrast, the strongly Bax-positive epithelial cells had no or only a narrow contact to the ductular lumen. The different patterns of Bax/Bcl-2 immunoreactivity in specific glandular epithelial cells suggest that there are also different grades of susceptibility towards apoptotic stimuli in individual glandular epithelial cells. We conclude that specific Bax/Bcl-2 expression patterns could reflect particular cell differentiation states, and that the strongly Bcl-2-positive cells in part could represent epithelial stem cells.     

Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.     

Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.     

Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst–matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.     

Expression of Fgf10 and Fgf receptors during development of the embryonic chicken stomach

Fibroblast growth factor 10 (FGF10) is involved in numerous different aspects of embryonic development and especially in active epithelial-mesenchymal interactions during morphogenesis of many organs as a mesenchymal regulator by activating its receptors (FGFR1b and FGFR2b) expressed in the epithelial tissue. FGFR2b is also activated by FGF7 although FGF7 does not bind to FGFR1b. To provide basic data to analyze function of FGFs in the developing gut, here we cloned Fgf7 and studied expression patterns of Fgf7, Fgf10 and Fgfr1-4 during the development of chicken stomach (glandular stomach; proventriculus and muscular stomach; gizzard). Fgf10 is expressed both in the proventricular and gizzard mesenchyme while Fgf7 is expressed only in gizzard mesenchyme. Fgfr1-4 are expressed both in the epithelium and mesenchyme with a different spatial expression patterns. Furthermore, RT-PCR analysis reveals that Fgfr1b and Fgfr2b are expressed only in epithelia of both organs.     

In vitro analysis of mesenchymal influences on the differentiation of stomach epithelial cells of the chicken embryo

It is well established that epithelial-mesenchymal interactions play important roles in the differentiation of stomach epithelial cells in the chicken embryo. To analyze mesenchymal influences on the differentiation of the epithelial cells, we developed a tissue culture system for stomach (proventriculus and gizzard) epithelia of chicken embryo, and examined their differentiation in the presence or absence of mesenchyme. Stomach epithelium from 6-day chicken embryo did not express embryonic chicken pepsinogen (ECPg), a marker molecule of glandular epithelial cells of proventriculus, while it expressed marker molecules of epithelial cells of the luminal surface of stomach, when cultured alone on the Millipore filter, covered with the gel consisting of extracellular matrix components. When the epithelium was recombined with mesenchyme separated by the filter, differentiation of the epithelium was affected by the recombined mesenchyme. Proventricular and lung mesenchymes induced the expression of ECPg in epithelial cells, and the expression was extensive when the gel contained basement membrane components. Proventricular and gizzard epithelia showed different responses to the mesenchymal action. We tested the effects of some growth factors on the differentiation of epithelial cells using this culture system. Furthermore we devised a "conditioned semi-solid medium experiment" for analysis of the inductive properties of proventricular and lung mesenchymes. The results of this experiment clearly demonstrated for the first time that diffusible factors from mesenchyme induce the differentiation of glandular epithelial cells in the absence of mesenchymal cells.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.