Localization of transferrin and transferrin receptors in rat testes

One of the major proteins secreted by rat Sertoli cells in culture is a transferrin-like protein (Skinner and Griswold, 1980). The purpose of this study was to quantitate the amount of testicular transferrin in fluids isolated from the testis by the use of a radioimmunoassay and to determine the location of transferrin and transferrin receptors in the testis by indirect immunofluorescence. Seminiferous tubule fluid, rete testis fluid, and testicular lymph were collected from rat testes and were found to contain 141 micrograms, 47 micrograms and 3.7 mg transferrin per ml of fluid, respectively. Serum was found to contain 3.7 mg/ml transferrin. Paraffin sections of rat testis were incubated with rabbit anti-rat transferrin, biotinylated goat anti-rabbit and fluorescein-conjugated avidin. Immunoreactive transferrin was thus localized on the proacrosome and nuclear cap of developing spermatids. Late spermatids showed transferrin over the entire region of the head but mature testicular spermatozoa exhibited little fluorescence. The interstitial tissue between seminiferous tubules fluoresced brightly, indicating a large amount of transferrin in this area. By pretreating sections with rat transferrin, the receptor for the protein was localized on and in spermatocytes and early round spermatids. Dividing germ cells were brightly fluorescent.     

Localization of growth hormone receptors in rat and human thyroid cells

Growth hormone (GH) exerts its multiple actions by binding to a specific receptor (GHR) widely distributed in the organism. It is well established that, in acromegaly, the thyroid gland is larger than normal and that GH increases triiodothyronin concentrations and decreases those of tetraiodothyronin (thyroxine). The aim of the present study was to analyze the presence of GHR and its mRNA in rat and human thyroid gland by Western blot, in situ hybridization techniques, and immunohistochemistry. A band of the expected size for GHR was shown in rat and human thyroid by Western blot. GHR immunoreactivity was found in virtually all follicles. The signal was mainly localized in the cytoplasm, although a nuclear positivity was also found. In situ hybridization techniques demonstrated the presence of GHR messenger RNA in the thyroid gland (cytoplasm of the follicular cells). These results provide direct morphological evidence that GHR is localized in the thyroid gland of mammals and opens up the possibility that GH regulates thyroid cell function directly or via local autocrine or paracrine production of insulin-like growth factor I.     

Localization of the site of human transferrin interacting with cellular receptors using monoclonal antibodies]

Antigenic determinants of the human transferrin molecule on the sublobe and lobe levels were localized for 7 monoclonal antibodies. Antibodies used have different effects on the interaction of the transferrin with its receptor. It was concluded that transferrin-receptor recognition was determined by NH2-lobe, the N2-sublobe playing major part. Dimerization of the transferrin molecules in solution was detected. Using the panel of monoclonal antibodies it was shown that dimerization accomplished by means of the COOH-lobes of transferrin molecules, the sites of interaction of the NH2-lobe with receptor being exposed. A model of the transferrin - receptor complex is proposed.     

Trophoblast transferrin and transferrin receptors in the host--parasite relationship of human pregnancy.

Transferrin and specific transferrin receptors are demonstrated on the microvillous surface of syncytiotrophoblast in human immature and term placentae by immuno histological techniques with the use of light and electron microscopy. That the distribution of transferrin is limited to the materno-foetal interface supports the hypothesis that binding of maternal transferrin to trophoblast receptors is involved in the process of iron transport to the foetus. Parallel studies with baboon placentae demonstrate the presence of trophoblast receptors which bind both baboon and human transferrin, thereby putting forward an experimental model which might be used to test the biological significance of placental transferrin receptors in primates. In addition, investigation of a large number of human cell lines shows that many transformed cells, but no normal cells (such as blood lymphocytes) or cells from primary culture (such as neonatal foreskin fibroblasts), possess the ability to bind transferrin to their membranes. These findings suggest that transferrin receptors may play important biological roles in addition to that of iron transport from mother to foetus. One such role could be the limitation of iron in intervillous spaces, thus depriving iron-requiring microorganisms of iron, hence serving as a non-specific factor of resistance for placentae. Another role for foetal transferrin receptors on trophoblasts could be to bind maternal transferrin at the materno-foetal interface, thus frustrating maternal immunosurveillance. This is similar to a mechahism used by schistosomes in the host-parasite relation where host proteins are bound by the parasite to escape immunological recognition. The presence of transferrin receptors on transformed cells suggests that this mechanism might also be employed by tumour cells. Finally, in view of previous studies which show that transferrin is required by stimulated lymphocytes to pass from the G1 to the S phase of cellular replication, it is proposed that trophoblast transferrin receptors could limit the amount of transferrin in intervillous spaces and thus impede the proliferation and possible cytotoxicity of maternal activated lymphocytes at the materno-foetal interface.     

"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Some molecular properties of human seminal transferrin (HSmT) in comparison with human serum transferrin (HSrT)

In this paper we report some structural features of human seminal transferrin (HSmT) in comparison with the homologous protein purified from human serum (HSrT). In particular, the sequence of the first 13 N-terminal amino acids of HSmT shows 12/13 of identity with the first 13 N-terminal amino acids of HSrT, the ninth residue of the former protein being not definitely determined. Moreover, HSrT and HSmT analysed under the same conditions, by means of reversed phase HPLC, thiol groups determination and second derivative spectroscopy, show a different content of amino acids. In particular, HSmT exhibits mainly: i) a lower Asx/Glx ratio; ii) a reduction of about 50% in Cys residues; iii) a decrease of Tyr and Trp residues. Eventually oligosaccharide parallel analyses of HSmT and HSrT show the same glycosidic bond and almost the same sugar content (around 5.5% w/w); conversely, HSmT lacks of sialic acid residues and probably it contains fucose. These results, taken all together, could be sound of interest to a better understanding of the possible physiological roles of HSmT.     

Regulation of transferrin receptors in human hematopoietic cell lines

Cells grown in the presence of ferric ammonium citrate or hemin exhibited a concentration and time-dependent decrease in 125I-transferrin (Trf) binding. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid (an iron chelator) exhibited a marked increase in Trf binding. The decrease or increase in binding activity observed under these different conditions of culture reflected, respectively, a reduction or increase in receptor number rather than an alteration in ligand receptor affinity. Growth of the cells in the presence of saturating concentrations of apotransferrin only induced a slight reduction in receptor number. Investigation of the Trf receptors' turnover and biosynthesis clearly showed that iron and hemin decreased the synthesis of Trf receptors without any modification of the receptor turnover; in contrast, protoporphyrin IX and picolinic acid markedly increased the synthesis of Trf receptors. Our results suggest that hemin, iron, and protoporphyrin IX may represent the main molecules involved in the regulation of Trf receptors.     

In vitro study on release of thyroid hormone in solitary autonomously functioning thyroid nodules using cell culture method

In vitro release of thyroid hormone was investigated under basal and TSH-stimulated conditions in the solitary autonomously functioning thyroid nodules (AFTN). A small portion (0.5 g of wet weight) of the nodules and adjacent thyroid tissues removed surgically from five patients with solitary AFTN were prepared for the dispersed cell culture. In the experiment on non TSH-stimulated (basal) conditions, those culture media which were totally replaced on the 5th day after primary culture were utilized for the determination of thyroxine (T4) and triiodothyronine (T3) by radioimmunoassay. T4 and T3 levels in culture media of the functioning nodules were 1.15 +/- 0.33 microgram/dl (mean +/- SEM) and 2.72 +/- 0.68 ng/ml, contrasted with levels of 0.67 +/- 0.09 microgram/dl and 1.24 +/- 0.22 ng/ml in the paranodular tissues. The mean ratios of T3/T4 of the nodules and paranodular tissues were 0.25 +/- 0.02 and 0.19 +/- 0.02, respectively (p less than 0.05). Meanwhile, in another experiment under TSH stimulatory conditions employing 40 and 80 microU/ml of human TSH, there were no significant differences in T4 and T3 releases when the two groups were compared.     

Loss and reappearance of transferrin receptors in human leukemic cell lines

Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation.     

Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae

The expression of human transferrin and lactoferrin binding activity in Haemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in all H. influenzae isolates tested but not detected in other Haemophilus species or in species of Pasteurella or Actinobacillus. Lactoferrin binding activity was only detected in 1/15 H. influenzae isolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron-loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron-loaded lactoferrin and apolactoferrin.     

Growth factor receptors gene expression and Akt phosphorylation in benign human thyroid nodules are unaffected by chronic thyrotropin suppression

Levothyroxine (L-T4)-based suppression of thyrotropin (TSH) secretion is widely used to prevent the growth of benign thyroid nodules, although the effectiveness of this approach has been demonstrated only in a subset of patients. In this study, we analyzed the in vivo effects of L-T4-mediated TSH suppression on elements of insulin/IGF-1-dependent growth-regulating pathways in tissues from patients with benign thyroid nodules. Nodular and non-nodular tissue specimens were collected from 63 patients undergoing thyroidectomy. 32 had received preoperative TSH suppressive therapy with TSH levels consistently below 0.5 mU/l (L-T4 group). TSH suppression had not been used in the other 31, and their TSH levels were normal (0.8-4 mU/l (control group). Quantitative RT-PCR was used to measure mRNA levels for TSH receptor, IGF1, IGF-1 receptor, insulin receptor, insulin receptor substrate 1 in nodular and non-nodular tissues from the 2 groups. Akt and phosphorylated Akt protein levels were detected by Western blot. Mean levels of mRNA for all genes tested were similar in the 2 groups, in both nodular and non-nodular tissues. The 2 groups were also similar in terms of phosphorylated Akt protein levels (measured by densitometric scan in 10 randomly selected nodules from each group). This is the first demonstration based on the study of human thyroid tissues that TSH suppression does not affect the expression of components of the insulin/IGF-1-dependent signaling pathways regulating thyrocyte growth. This may explain the lack of effectiveness of TSH-suppressive therapy in a substantial percentage of benign thyroid nodules.     

Localization of sex steroid receptors in human skin

Sex steroid hormones are involved in regulation of skin development and functions as well as in some skin pathological events. To determine the sites of action of estrogens, androgens and progestins, studies have been performed during the recent years to accurately localize receptors for each steroid hormone in human skin. Androgen receptors (AR) have been localized in most keratinocytes in epidermis. In the dermis, AR was detected in about 10% of fibroblasts. In sebaceous glands, AR was observed in both basal cells and sebocytes. In hair follicles, AR expression was restricted to dermal papillar cells. In eccrine sweat glands, only few secretory cells were observed to express AR. Estrogen receptor (ER) alpha was poorly expressing, being restricted to sebocytes. In contrast, ERbeta was found to be highly expressed in the epidermis, sebaceous glands (basal cells and sebocytes) and eccrine sweat glands. In the hair follicle, ERbeta is widely expressed with strong nuclear staining in dermal papilla cells, inner sheath cells, matrix cells and outer sheath cells including the buldge region. Progesterone receptors (PR) staining was found in nuclei of some keratinocytes and in nuclei of basal cells and sebocytes in sebaceous glands. PR nuclear staining was also observed in dermal papilla cells of hair follicles and in eccrine sweat glands. This information on the differential localization of sex steroid receptors in human skin should be of great help for future investigation on the specific role of each steroid on skin and its appendages.     

The region of human transferrin involved in binding to bacterial transferrin receptors is iocalized in the C-lobe

Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin.     

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley-Liss, Inc.     

Characterization of transferrin receptors on plasma membranes of lactating rat mammary tissue

Little is known about the transport of iron into the mammary secretory cell and the process of milk iron secretion. The concentration of iron in milk is remarkably unaffected by maternal iron status, suggesting that the uptake of iron into the mammary gland is regulated. It is known that iron enters other cells via transferrin receptor-mediated endocytosis. This study was designed to isolate and characterize the mammary gland transferrin receptor in lactating rat mammary tissue using immunochemical techniques. The existence of functional mammary gland transferrin receptors in lactating rodents was demonstrated using radiolabel-binding techniques. Isolation of mammary transferrin receptors by affinity chromatography was confirmed using immunoelectrophoresis and slot blot analysis. The intact transferrin receptor was found to have a molecular weight of 176 kd as determined by Western blotting followed by scanning densitometry. Reduction of the receptor with beta-mercaptoethanol gave a molecular weight of 98 kd. An additional immunoreactive band of 135 kd was observed. The presence of transferrin receptors in normal lactating rat mammary tissue is likely to explain iron transport into mammary tissue for both cellular metabolism and milk iron secretion.     

Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.