Purification of desmoglein II: a method for the preparation and fractionation of desmosomal components.

We have developed rapid and efficient methods for the isolation of desmosomes and the fractionation of their components. These methods involve the use of 6 M guanidine HCl to isolate the desmosomes from bovine epidermis, followed by hydroxyapatite column chromatography in the presence of SDS to fractionate the desmosomal components. All of the desmoplakins and desmogleins were purified at least partially by these procedures, and desmoglein II was purified to apparent homogeneity. We expect these procedures to facilitate a detailed biochemical analysis of the molecular components of desmosomes. In addition, these methods may be applicable to the purification of other plasma membrane domains involved in cell adhesion.

Identification of a conserved region common to cadherins and influenza strain A hemagglutinins.

Cadherins are a family of integral membrane glycoproteins that mediate homophilic, calcium-dependent cell adhesion in vertebrate species. The primary structures of six members of the cadherin family have recently been determined. The extracellular portion of these proteins is composed of five domains, the first of which is the most highly conserved among cadherins. Previous searches of protein sequence databases have revealed little or no sequence homology between cadherins and other proteins. Here we report that the first extracellular domain of cadherins exhibits substantial sequence homology with the amino termini of influenza strain A hemagglutinins. These regions of sequence homology have been shown to be functionally important in both cadherins and hemagglutinins. Our observations suggest that a functional domain of cadherins is conserved among other proteins.

Identification and developmental regulation of cadherin messenger ribonucleic acids in the rat testis.

Cellular interactions in the rat testis are suggested by the presence of gap junctions between developing germ cells and Sertoli cells as well as tight junctions between adjacent Sertoli cells. Cadherins are cell surface proteins that mediate calcium-dependent intercellular adhesion. In these experiments the presence and developmental regulation of three cadherins have been examined: epithelial cadherin (E-Cad), neural cadherin (N-Cad), and placental cadherin (P-Cad). Northern blot analysis of testicular RNA indicates the presence of N-Cad [4.3 and 3.5 kilobases (kb)] and P-Cad (3.5 kb) transcripts. No E-Cad message was detected. To determine whether mRNA concentrations for P-Cad and N-Cad are regulated during postnatal rat testicular development, testes from rats ranging in age from 7-91 days were subjected to Northern blot analysis. Relative P-Cad mRNA levels were highest at 7 days of age and decreased to almost half of these levels by day 14. P-Cad mRNA levels subsequently decreased to low levels and remained constant thereafter. This contrasted with the developmental pattern observed for the 4.3-kb N-Cad transcript, which was low early in testicular development but increased to peak levels on day 42, coincident with the shedding of the first sperm. N-Cad mRNA concentrations decreased from 42 to 56 days and then remained constant until 91 days. While mouse P-Cad antibody did not cross-react with rat P-Cad, immunoblots of testicular membrane protein preparations identified the presence of immunoreactive N-Cad protein in the testis. The presence of N-Cad protein confirms that N-Cad mRNA is translated in this tissue. The developmental patterns of P-Cad and N-Cad mRNA suggest a role for P-Cad early in testicular development, while N-Cad appears to play a role in later stages of spermatogenesis.

Cellular heterogeneity in the membrana granulosa of developing rat follicles: assessment by flow cytometry and lectin binding.

The hormone-mediated maturation of ovarian follicles is apparently accompanied by position-specific differentiation of cells of the membrana granulosa. We have assessed the extent of this cellular heterogeneity by flow cytometry using a variety of fluorescein isothiocyanate-labeled lectins as probes. Follicular development was stimulated in immature rats by treatment with either diethylstilbestrol (DES) or equine CG (eCG). Lectin binding to monodispersed rat granulosa cells was then analyzed by flow cytometry. Our results demonstrate that there are two distinct populations of small (4-7 microM) and large (9-12 microM) granulosa cells in follicles from DES- and eCG-treated animals. Both populations appear to be mitotically active and show specific lectin-binding characteristics. Six lectins (canavalia ensiforms, triticum vulgaris, maclura pomifera, erythrina cristagalli, jacalin, and vicia villosa) bind equally to both small and large granulosa cells from the DES- and eCG-treated rats. In contrast, no binding to either cell population was detected with six other lectins (dolichos biflorus, griffonia simplicifolia-II, lycopersicon esculentum, datura stramonium, solanum tuberosum, and ulex europaeus). Furthermore, four galactose-binding lectins (bauhinia purpurea, glysine maximus, griffonia simplicifolia-I, and arachis hypogaea) were found to identify specific subsets of granulosa cells. Three of these lectins (bauhinia purpurea, glysine maximus, and griffonia simplicifolia-I) bind to only small granulosa cells from either DES- or eCG- treated immature rats. The fourth lectin (arachis hypogaea) identifies subpopulations of both small and large granulosa cells. Application of the four galactose-specific lectins to fixed sections of frozen ovaries demonstrated binding to the perioocyte and cumulus granulosa cells. We conclude that cellular heterogeneity exists within the follicular epithelium at various stages-specific lectin-binding sites.

Estrogens potentiate the stimulatory effects of follicle-stimulating hormone on N-cadherin messenger ribonucleic acid levels in cultured mouse Sertoli cells.

Gonadal steroids and FSH are key regulators of Sertoli cell function. N-Cadherin (N-cad) is a calcium-dependent cell adhesion molecule that mediates Sertoli cell-germ cell interactions. We recently demonstrated that steroids, in particular estradiol, are potent regulators of testicular N-cad messenger RNA (mRNA) levels in vivo. In view of the cooperative effects of steroids and FSH on Sertoli cell-germ cell interactions, we examined the combined effects of these hormones on N-cad mRNA levels in cultured mouse Sertoli cells. FSH was capable of increasing N-cad mRNA levels 2-fold in these cells. The effects of FSH on N-cad mRNA levels in cultured Sertoli cells were mimicked by cAMP-inducing agents. Treatment of the Sertoli cell cultures with FSH and estradiol stimulated N-cad mRNA levels 3-fold, whereas steroids alone had no effect on N-cad mRNA levels. These studies demonstrate that FSH and estradiol in combination are required to achieve maximal N-cad mRNA levels in cultured Sertoli cells. The results obtained from these studies substantiate the hypothesis that estrogens play a pivotal role in regulating spermatogenesis.

Distribution and regulation of epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin in the rat epididymis.

The epithelium of the epididymis possesses an elaborate network of tight junctions between principal cells which is altered as a function of postnatal age. Cadherins are implicated in the formation of tight junctions. The objective of the present study was to determine whether RNA transcripts for cadherins were present in the epididymis, and if so, how they were hormonally regulated. Using specific cDNA probes for epithelial cadherin (E-Cad) and neural cadherin (N-Cad), Northern blot analysis was used to study steady state levels of cadherin mRNAs. A major E-Cad mRNA species of 4.7 kilobases and a weaker 4.3-kilobase species were observed in the epididymis. No signal for N-Cad was detected. Steady state mRNA levels for E-Cad were highest in the caput and corpus epididymidis and were almost 4 times higher than those in the initial segments and cauda epididymidis; no signal was detected in the vas deferens. Light microscopic immunocytochemical localization of E-Cad revealed a reaction over the principal cells of the entire epididymis. The relative intensities of the immunoreactivity suggested that the E-Cad protein concentration was highest in the corpus, followed by the caput, cauda, and initial segments of the epididymis. There was no reaction over the epithelial basal and clear cells or intraepithelial halo cells. Three days after bilateral orchidectomy, E-cad mRNA was decreased by 75% in the caput epididymidis. A dose-dependent maintenance of mRNA concentration for E-Cad was observed throughout the epididymis of orchidectomized rats after replacement with testosterone. Fourteen days after unilateral orchidectomy, no differences were observed in the concentrations of epididymal E-Cad mRNA between control and unilaterally orchidectomized rats. Together, these data demonstrate that mRNA for E-Cad is present and translated in the rat epididymis, is differentially distributed along this tissue, and can be regulated by circulating androgens.

Cadherins and cadherin-associated molecules in the developing and maturing rat testis.

The calcium-dependent class of cell adhesion molecules known as cadherins mediate homotypic cell interactions in most epithelia. We have now investigated the expression and distribution of cadherins and cadherin-associated molecules in the developing and maturing rat testis. E-Cadherin was not detected in the seminiferous tubule at any time in development or in the adult. In contrast, Leydig cells expressed E-cadherin between day 15 of gestation and postnatal day 3. alpha- and beta-catenins were expressed throughout the developing testis, but were particularly prominent in Leydig cells. In the maturing testis, alpha-catenin and plakoglobin became progressively more restricted to the basal part of the seminiferous epithelium and by 23 days exhibited a pattern characteristic of the Sertoli cell junctional complex. beta-Catenin recruitment to the Sertoli cell junctional complex was not complete until 60 days. alpha-Catenin and plakoglobin were not present at sites of Sertoli cell-germ cell contacts. Northern blot analysis of testicular RNA showed three mRNA species hybridizing with N-cadherin cDNA. A pan-cadherin antibody specific for a region of the highly conserved C-terminal of all cadherins stained sites of Sertoli-spermatocyte and Sertoli-round spermatid contact in the adult rat seminiferous epithelium, but did not stain the Sertoli cell tight junctional complex. Western blots of testicular extracts indicated that the molecule(s) recognized by these antibodies had an approximate molecular mass of 120 kilodalton, typical of members of the cadherin family. Therefore, although Sertoli cells do not express E-cadherin, another member(s) of the cadherin family is present in the testis, but may not be directly involved in tight junction dynamics as in other cells. Instead, cadherin-mediated adhesion is likely to be involved in Sertoli cell-germ cell interactions. As catenins are not present at these sites, our results suggest a catenin-independent role of cadherins in germ cell adhesion to Sertoli cells.

Estradiol regulates E-cadherin mRNA levels in the surface epithelium of the mouse ovary.

E-cadherin is a calcium-dependent cell adhesion molecule which is present in the surface epithelium of the mouse ovary. This cell adhesion molecule has been implicated as a suppressor of tumorigenesis. The regulators of E-cadherin mRNA levels in the ovary have not been identified. We have examined the ability of steroids to influence ovarian E-cadherin mRNA levels in vivo. Immature mice were injected with either progesterone, testosterone, dihydrotestosterone, 17-beta estradiol or 17-alpha estradiol. Only 17-beta estradiol caused a rapid and significant increase in the ovarian E-cadherin mRNA levels. We speculate that this steroid is a key regulator of E-cadherin-mediated epithelial cell interactions in vivo. We also discuss the possibility that the carcinogenic effects of estrogens on the ovary may be related to their ability to regulate E-cadherin levels in this tissue.

The loss of E-cadherin mRNA transcripts in rat prostatic tumors is accompanied by increased expression of mRNA transcripts encoding fibronectin and its receptor.

We compared the levels of mRNA transcripts encoding E-cadherin, N-cadherin, beta 1 integrin subunit, alpha 5 integrin subunit and fibronectin in the normal rat prostate gland, as well as in tumors derived from three invasive sublines (G, MatLyLu, AT-2) of the Dunning R-3227 rat prostatic adenocarcinoma. E-cadherin mRNA transcripts were only detectable in total RNA extracts prepared from normal rat prostates, whereas N-cadherin mRNA transcripts were only found in normal rat brains. In contrast, the mRNA transcripts encoding the beta 1 integrin subunit, alpha 5 integrin subunit and fibronectin were all elevated in the tumors, as compared to the levels of these transcripts in normal tissues. Our results suggest that there is an inverse correlation between cadherin and integrin mRNA levels in rat prostatic tumors.

Hormonal regulation of N-cadherin mRNA levels in rat granulosa cells.

We have examined the ability of hormones to modulate the steady-state levels of N-cadherin mRNA transcripts in aggregated and dispersed rat granulosa cell populations. Estradiol and follicle-stimulating hormone (FSH) had no effect on the levels of N-cadherin mRNA transcripts in aggregated granulosa cells. In contrast, these two hormones stimulated N-cadherin mRNA levels in dispersed granulosa cells. This is the first report that estradiol and FSH are capable of regulating N-cadherin mRNA levels. The results also suggest that the N-cadherin mRNA levels in dispersed and aggregated granulosa cells are regulated by different mechanisms.

Isoelectric forms of clusterin isolated from ram rete testis fluid and from secretions of primary cultures of ram and rat Sertoli-cell-enriched preparations.

Clusterin, a cell aggregating factor isolated from ram rete testis fluid (RTF), is shown to contain 14.7% hexoses, 13.6% glucosamine, and 7.9% sialic acid. The isoelectric point (pI) of the predominant electrophoretic form of clusterin from ram RTF is 3.7. After treatment with neuraminidase, the pI values become more basic, with the majority of the material being eluted from a chromatofocusing column at pH values between 4.9 and 5.1. Intact clusterin binds quantitatively to wheat germ agglutinin - Sepharose 6 MB, but after treatment with neuraminidase only 49% specifically binds. Clusterin isolated from proteins secreted by primary cultures of ram Sertoli-cell-enriched preparations was shown to have properties similar to those of intact clusterin isolated from ram RTF. In contrast, clusterin isolated from proteins secreted by primary cultures of rat Sertoli- or granulosa-cell-enriched preparations has isoelectric forms which more closely resemble those of neuraminidase-treated ram clusterin.

Estradiol stimulates cadherin expression in rat granulosa cells.

We have investigated the ability of hormones to modulate cadherin expression by differentiating cells. Immunocytochemical and immunoblot techniques were employed to analyze the effects of estradiol and follicle-stimulating hormone on cadherin expression in rat granulosa cells. Estradiol was shown to stimulate the expression of cadherin by these cells. This is the first report of a hormone regulating the levels of cadherin in differentiating cells.

Cadherins as modulators of angiogenesis and the structural integrity of blood vessels.

The endothelial cell adhesion molecules, neural- and vascular endothelial-cadherin play essential roles in the formation of stable and fully functional blood vessels. This commentary discusses the multiple functions of these two cadherins in angiogenesis and the maintenance of blood vessel structural integrity.

A comprehensive survey of the cadherins expressed in the testes of fetal, immature, and adult mice utilizing the polymerase chain reaction.

The cadherins are a family of calcium-binding membrane glycoproteins. Most cadherins are capable of acting as cell adhesion molecules (CAMs). In order to begin a thorough analysis of the roles of these CAMs in the testis, we employed a reverse transcriptase-polymerase chain reaction (RT-PCR) strategy to identify the cadherins expressed in this tissue at various stages of development. Oligonucleotides encoding amino acid sequences that are conserved among all of the known cadherins were used as primers in the RT-PCR, with cDNA preparations of fetal, newborn, 7-day, 21-day, and adult mouse testes employed as templates. The PCR products were subcloned into a plasmid vector and sequenced. On the basis of the nucleotide sequences of these PCR products, we have determined that five previously characterized cadherins (E-cadherin, N-cadherin, P-cadherin, K-cadherin, and OB-cadherin), as well as two novel cadherins (T1-cadherin and T2-cadherin), are expressed at various stages during testicular development. In order to determine the expression patterns of these cadherins, we ascertained the mRNA levels of each cadherin normalized to the levels of hypoxanthine phosphoribosyltransferase mRNA in fetal, newborn, 7-day, 21-day, and adult mouse testes. We observed that N-cadherin mRNA is expressed at all stages of testicular development, with maximal levels being present in the testes of 21-day-old mice. Furthermore, we found that E-, P-, K-, OB-, and T2-cadherin mRNAs are all expressed in the fetal gonad. The testicular levels of these cadherin mRNAs decreased dramatically after birth. Conversely, T1-cadherin mRNA was not detected in the fetal, newborn, and 7-day-old testes but was present in 21-day-old and adult testes. T1-cadherin levels were 10-fold higher in the testes of adult mice, compared to the levels found in the testes of 21-day-old mice. We speculate that these cadherins will be found to be intimately involved in mediating cell interactions during testicular development.

Developmental expression and distribution of N- and E-cadherin in the rat ovary.

The calcium-dependent cell adhesion molecules, cadherins, regulate intercellular junction formation, cell sorting, and the establishment of cell polarity. Their important role in tissue remodeling suggests an involvement in ovarian cellular rearrangements throughout postnatal development. The ovary has a complex topology, and the ovarian follicle undergoes significant cellular rearrangements during its development. Cadherins have been detected previously in whole ovaries and in ovarian cells and cell lines with some immunolocalization in fetal and adult ovaries. This study examines the expression and localization of N- and E-cadherin throughout prepubertal ovarian and follicular development in the rat. We analyzed ovarian cadherin expression in rats from Day 19-20 of gestation to 25 days postpartum, during which follicle formation and folliculogenesis are the dominant ovarian events. Reverse transcriptase polymerase chain reaction detected N- and E-cadherin mRNA expression in the ovaries at all the ages examined. Semiquantification of Western blots of whole ovary extracts confirmed the presence of ovarian N- and E-cadherin protein at all ages with both showing peak expression at 7 days of age. Immunostaining revealed N- and E-cadherin expression in follicular and extrafollicular cell types, but only E-cadherin showed follicle-stage-dependent expression. The changes in cadherin expression, concurrent with ovarian growth and folliculogenesis, suggest a function for cadherins in the morphological and functional development of the prepubertal rat ovary.

An N-cadherin-like protein contributes to solute barrier maintenance in cultured endothelium.

We investigated the role of cadherins in the solute barrier maintained by endothelial cells in vitro. Cell-column chromatographic measurement of endothelial barrier showed that reducing normal extracellular calcium from 1.2 to 0.12 mM increased endothelial permeability to 250% of baseline after 20 min. Restoring normal calcium restored the barrier within 15 min which remained stable for at least 60 min. We used sulfo-NHS-biotin and anti-cadherin antibodies to characterize endothelial proteins with possible roles in the maintenance of endothelial barrier. The non-specific probe sulfo-NHS-biotin identified at least ten endothelial cell surface proteins, with greatest labelling occurring at molecular weights of 125 and 145 kD. Six proteins, including the 125 and 145 kD proteins, associated with the cytoskeleton. Western blotting for the presence of classical cadherins containing the conserved cytoplasmic sequence CDPTAPPYDSLLVFDYEG detected two bands at 145 and 125 kD which associated with the cytoskeleton. Western blotting with an antibody, which recognizes FHLRAHAVDINGNQV, an extracellular homotypic binding region of N-cadherin, detects three bands. Of these three, one protein had a molecular weight of 125 kD and was associated with the cytoskeleton. Immunofluorescence with both N-cadherin and anti-peptide 1 antibodies found staining at endothelial cell borders. The utility of a newly developed cell-column calcium switch assay was tested by verifying the functional role of the previously described epithelial cadherin, uvomorulin, in epithelial barrier. We then applied this method to endothelial cell columns and found the N-cadherin antibody interfered with the reforming of interendothelial junctions. These results suggest that, as in epithelial cells, cadherins in bovine endothelial cells have a functional role in forming the calcium sensitive endothelial junction and may play an important role in the formation of normal barrier.

Studies on hexosaminidase forms in Chinese hamster ovary cells.

The hexosaminidase forms present in Chinese hamster ovary cells grown in culture were partially purified and their kinetic and physical properties were characterized. The cells were found to contain three hexosaminidase forms, which could be resolved by ion-exchange chromatography. These three forms, designated hexosaminidases I, II and III, had properties similar to those of human hexosaminidases A, B and C, respectively. Hexosaminidase III, like human hexosaminidase C, appears to be regulated by a different mechanism than the other two activities. The data presented in this report show that Chinese hamster ovary cells are an excellent source of enzyme activity for investigating the complex properties of the various mammalian hexosaminidase forms.

Properties of hexosaminidases in cell-free extracts of rainbow trout livers and effects of thyroid hormones.

1. The kinetic and physical properties of hexosaminidase (EC 3.2.1.30) activity were studied in liver extracts of juvenile rainbow trout. 2. Differential centrifugation studies failed to unequivocally demonstrate the sub-cellular localization of hexosaminidase, but indicated that the hexosaminidase activity was associated with the mitochondrial and lysosomal fractions. 3. Cell-free, Triton X-100 liver extracts indicated a pH optimum and activation energies comparable to those of mammals, but for other properties trout hepatic hexosaminidase differed markedly from its mammalian counterpart. 4. In contrast to the situation in rats, trout hexosaminidase was uninfluenced by a wide range of T4 and T3 doses administered by either injection or immersion routes. 5. T4 administration increased plasma T4 with minor change in plasma T3, indicating inhibition of T4 to T3 conversion by high T4 levels.

Cadherins, steroids and cancer.

The cadherins are a family of calcium-dependent cell adhesion molecules which are thought to be key regulators of morphogenesis. This review contains a discussion of the structure, function and regulation of these cell adhesion molecules. In particular, we discuss recent studies that demonstrate the ability of steroids to modulate cadherin levelsin vivo. We speculate that steroids and estrogenic organochlorines exert their diverse morphoregulatory actions on tissues by altering cadherin levels.

Developmental regulation of a cadherin during the differentiation of skeletal myoblasts.

Cadherins are a family of integral membrane glycoproteins which mediate calcium-dependent intercellular adhesion in vertebrate species. Here we present evidence that fusion-competent rat L6 myoblasts express a cadherin (Mr 127 kDa). The levels of this cadherin were found to be developmentally regulated. Maximal levels were expressed prior to fusion. The increase in cadherin levels observed during differentiation was prevented by the differentiation inhibitor, 5-bromo-2'-deoxyuridine. L6 myoblasts grown in the presence of anti-cadherin antibodies exhibited an altered morphology in comparison to control cultures, coupled with decreased myoblast fusion. These data indicate that the developmental regulation of cadherin is part of the program of terminal differentiation of skeletal myoblasts, and that cadherins are involved in the process of myoblast fusion.