Beta 1C Research Articles

Regulation of β1C and β1A Integrin Expression in Prostate Carcinoma Cells

beta(1C) and beta(1A) integrins are two splice variants of the human beta(1) integrin subfamily that act as an inhibitor and a stimulator of cell proliferation, respectively. In neoplastic prostate epithelium, both these variants are down-regulated at the mRNA level, but only beta(1C) protein levels are reduced. We used an experimental model consisting of PNT1A, a normal immortalized prostate cell line, and LNCaP and PC-3, two prostate carcinoma cell lines, to investigate both the transcription/post-transcription and translation/post-translation processes of beta(1C) and beta(1A). Transcriptional regulation played the key role for the reduction in beta(1C) and beta(1A) mRNA expression in cancer cells, as beta(1C) and beta(1A) mRNA half-lives were comparable in normal and cancer cells. beta(1C) translation rate decreased in cancer cells in agreement with the decrease in mRNA levels, whereas beta(1A) translation rate increased more than 2-fold, despite the reduction in mRNA levels. Both beta(1C) and beta(1A) proteins were degraded more rapidly in cancer than in normal cells, and pulse-chase experiments showed that intermediates and/or rates of beta(1C) and beta(1A) protein maturation differ in cancer versus normal cells. Inhibition of either calpain- or lysosomal-mediated proteolysis increased both beta(1C) and beta(1A) protein levels, the former in normal but not in cancer cells and the latter in both cell types, albeit at a higher extent in cancer than in normal cells. Interestingly, inhibition of the ubiquitin proteolytic pathway increased expression of ubiquitinated beta(1C) protein without affecting beta(1A) protein levels in cancer cells. These results show that transcriptional, translational, and post-translational processes, the last involving the ubiquitin proteolytic pathway, contribute to the selective loss of beta(1C) integrin, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

β1C Integrin Expression in Human Endometrial Proliferative Diseases

Integrins are ubiquitous cell adhesion molecules that are involved in maintaining normal tissue morphology and have been implicated in the aggressive behavior of several malignancies. beta 1C integrin is an alternatively spliced variant of the beta 1A integrin subunit that, at variance with beta 1A, inhibits epithelial cell proliferation. beta 1C integrin is expressed in non-proliferative, benign prostatic epithelium and is down-regulated in prostatic adenocarcinoma. In the current study, we examined beta 1C expression at mRNA and protein levels in 18 endometrial adenocarcinoma and in 20 endometrial hyperplastic tissues, using Northern and Western blotting analysis and immunohistochemistry. The pattern of integrin expression was compared to that of the endometrium of 14 normal cycling women. The results of this study document inhibited beta 1C integrin expression in endometrial adenocarcinoma, both at the mRNA and protein levels, at variance with significantly up-regulated beta 1C mRNA expression in endometrial hyperplasia, in comparison with normal proliferative endometria. Our data suggest a key role of the regulation of beta 1C integrin expression in the pathogenesis of endometrial proliferative diseases: beta 1C integrin may act as growth modulator in cancer cells, playing a role in downstream intracellular signaling.

Differential expression of beta 1c integrin messenger ribonucleic acid and protein levels in human endometrium and decidua during the menstrual cycle and pregnancy.

beta(1C) and beta(1A) integrins are alternatively spliced variants of the human beta(1)-subunit; the former has been shown to inhibit cell proliferation, and the latter to promote it. Although some components of the beta(1) integrin subfamily are expressed in human endometrial and decidual cells during the menstrual cycle and early pregnancy, to date no information is available about the expression of beta(1C) integrin in endometrial and decidual tissues and its possible roles during implantation and pregnancy. To gain further insight on this subject, we have explored beta(1C) integrin expression in endometrial (proliferative, secretory, and atrophic) and decidual (from the first and third trimesters of pregnancy) tissue samples at both gene and protein levels by Northern and Western blotting analyses and by immunohistochemistry. beta(1A) protein levels were also measured in the same tissues as a control. The results of this study demonstrate that both beta(1C)- and beta(1A)-subunits are expressed in the endometrium and decidua. In the former, maximal beta(1C) expression was detected in atrophic endometria, whereas beta(1A) expression levels were increased in secretive and decreased in atrophic endometrial tissues compared with proliferative endometria. In addition, whereas beta(1A) levels were significantly increased in decidual tissues, compared with proliferative endometria, beta(1C) expression was dramatically reduced in the same tissues, thus pointing to selective down-regulation of beta(1C) expression in the decidua. These data suggest that the expression of beta(1C) integrin, a very efficient inhibitor of cell proliferation, may be modulated by the maternal microenvironment and may play a fundamental role in mediating trophoblast outgrowth and migration during pregnancy.

Down-Regulation of β 1C Integrin in Breast Carcinomas Correlates with High Proliferative Fraction, High Histological Grade, and Larger Size

beta(1C) integrin is an unspliced form of the integrin beta(1) subfamily, which has been shown to inhibit cell proliferation in vitro. Using an affinity-purified rabbit antibody, we have investigated 283 previously untreated breast carcinomas, with the aim of ascertaining the actual prevalence of beta(1C) expression in these tumors and of defining its pathological correlates. Immunoblotting and reverse transcriptase-polymerase chain reaction experiments have also been performed in selected cases, to confirm the immunocytochemical findings. Overall, beta(1C) immunoreactivity was down-regulated (ie, expressed in < 50% of the neoplastic cells) in 114 cases (40.3%). Down-regulation of beta(1C) expression in breast carcinomas correlated significantly with the tumor grade, the proliferative fraction (as evaluated by Ki-67 immunostaining with the MIB-1 monoclonal antibody), the estrogen and progesterone receptor status, and the tumor size (pT classification) and marginally with the node status. In a multivariate analysis with all available measures fitted simultaneously, tumor grade (P = 0.004), Ki-67 immunolabeling (P = 0.01), and pT categories (P = 0.04) were significantly associated with beta(1C) immunoreactivity. Although the short follow-up time (2-3 years) of the current series of patients does not allow the performance of survival analyses, the correlation of beta(1C) expression with tumor size, grade, and proliferative fraction and its alleged role as an upstream regulator of p27(kip1) make this integrin variant a likely novel prognostic parameter for invasive carcinomas of the breast.

Alternatively spliced variants: a new view of the integrin cytoplasmic domain.

A large number of studies have underscored a major role for the integrin alpha beta cytoplasmic domains in the modulation of cell functions. Cytoplasmic domain variants of the beta 1, beta 3, beta 4, alpha 3, alpha 6 and alpha 7 subunits have been described. These molecules are generated by alternative splicing events and are expressed in a cell- or tissue-type specific manner. Some of these variants (beta 1C, beta 1D, alpha 6A and alpha 7A) are predominantly expressed upon differentiation and have been shown to be regulated during development. The studies on the structure-function relationship of the integrin variant subunits, published between 1989 and now, will be reviewed here for the first time. The results demonstrate that differences in the cytoplasmic domain do not affect either the alpha beta heterodimer formation or the ligand specificity. Instead, alternatively spliced integrin cytoplasmic domains appear to be essential modulators of receptor localization, cell proliferation and migration, as well as phosphorylation of signaling molecules. These observations lead to the current hypothesis that cell-type specific regulation of alternatively spliced integrin cytoplasmic domains may provide a highly specialized mechanism to control cell growth and intracellular signaling pathways.

Modulation of cell proliferation by the integrin cytoplasmic domain

Integrin adhesion receptors modulate cell functions, including cell proliferation and survival. The beta 1C integrin, an alternatively spliced form of beta 1A, containing a unique cytoplasmic domain sequence, inhibits cell growth in vitro. In vivo, the expression of beta 1C correlates with a benign, nonproliferative phenotype in epithelial cells. The studies discussed in this article indicate that modulation of cell proliferation, in normal or pathological conditions, might be achieved by the regulated expression of variant integrin subunits.

The Novel Structural Motif Gln795–Gln802 in the Integrin β1C Cytoplasmic Domain Regulates Cell Proliferation

Alternative splicing of the integrin beta 1 subunit mRNA generates a variant form, beta 1C, with a unique cytoplasmic domain that differs from beta 1 for a 48-amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)1 cells transiently transfected with cDNAs coding for human integrin beta 1C or beta 1 subunits or mutants containing truncated forms of the beta 1C cytoplasmic domain. A differential effect of beta 1C and beta 1 on cell proliferation was observed. Expression of wild type beta 1 was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of beta 1C. Cells expressing the beta 1C mutant truncated at Leu794 and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three beta 1C deletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802 of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of beta 1C expression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing beta 1C, full-length or deletion mutants, or beta 1 equally adhered to a functionally inhibitory monoclonal antibody against human beta 1 integrin. Expression of beta 1C inversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of beta 1C was induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo.

Inhibition of cell cycle progression by the alternatively spliced integrin beta 1C.

Integrins regulate cell growth, differentiation, and behavior in many systems. Integrin beta 1C (beta 1S) is an alternatively spliced variant of integrin beta 1 with a specific cytoplasmic domain and is expressed in several human tissues. Human beta 1c transiently expressed in mouse 10T1/2 fibroblasts showed a diffuse pattern of cell surface staining, whereas beta1 localized to focal adhesions. Moderate concentrations of beta 1C had no effect on actin stress fibers or focal adhesions, but markedly inhibited DNA synthesis. Inhibition by beta 1C mapped to the late G1 phase of the cell cycle, near the G1-S boundary. Thus, alternative splicing of beta1 results in transmission of distinct signals that may regulate growth in vivo.

Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association.

Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.

Estimation of Postmortem Interval Based on the Third Component of Complement (C3) Cleavage

To estimate postmortem interval (PMI), the spontaneous conversion of the native third component of complement (C3) to its derived fragments in whole blood was studied by crossed immunoelectrophoresis. C3 cleavages in vitro at different temperatures showed that the incubation of whole blood at a higher temperature led to a faster conversion of beta 1C (native C3) to beta 1A (C3c). In cadaveric blood, we found a significant positive correlation between percentage of C3 cleavage and PMI. From these results, it is possible to estimate PMI from the ratios of C3 cleavage.

Measurement of fragments of the third component of human complement on erythrocytes by a new immunochemical method.

This paper describes a new antiglobulin consumption method to quantitate the fragments of the third component of human complement (C3) on red blood cell (RBC) membranes. Zymosan-bound C3, which can be stored frozen at -80 degrees C for years, was used as a standard in these tests. Using anti-C3c antibody, zymosan-bound C3 could be calibrated against soluble converted C3 (beta 1A), but not against soluble, native C3 (beta 1C). Calibration with several commercial serum standards yielded virtually identical values. Approximately 79.8 +/- 28.2 C3d molecules (mean +/- 1 SD, n = 50) were detected on normal, freshly collected RBC by this method, whereas no C3c fragments were noted. EC43, prepared by dilution of blood samples with low ionic strength solution, had between 650 and 3,100 C3 molecules/RBC when measured with anti-C3c and between 1,140 and 6,500 C3 molecules when measured with anti-C3d. These data indicated that part of the C3b molecules on EC43 had cleaved to C3d. EC43 are reported to have up to 200,000 C3 molecules when measured by other techniques. To resolve this discrepancy, EC43 were prepared by dialysis of blood samples against low ionic strength solution. About 97.5% of C3 remained in plasma after dialysis supporting the results of our tests. The new assay is an accurate and sensitive method of quantitating C3 fragments bound to RBC in vivo and in vitro.

Studies on the binding of complement factor C3 to the surface of human blood platelets.

Employing immunofluorescent staining methods, platelets from several adult patients with chronic idiopathic thrombocytopenic purpura, with systemic lupus erythematosus, and with classical rheumatoid arthritis stained positively with antisera to C3(beta 1C) and C3dg(alpha 2D). In some cases, platelet-bound C3dg antigens, but no evidence of other C3 antigens were observed. Platelets that were positive for C3 and/or C3dg were always positive for IgG or for both IgG and IgM. Platelets from normal subjects stained negatively for C3, C3dg, IgG, and IgM. Furthermore, various incubation experiments were performed to investigate the uptake of C3 and its subfragments on normal platelets. Positive platelet staining for C3 was obtained after incubation with normal and C3b-containing sera, but not after incubation with native C3. Platelets incubated with C3c(beta 1A)/C3dg-containing serum gave a positive staining for C3dg, but not for C3c. None of the sera stained positively for IgG or IgM. Thus, in contrast to platelets from several of the patients, no in vivo binding of C3 antigens on normal platelets could be established. However, in vitro they could take up both C3b and C3dg, apparently by specific binding to platelet surface structures.

Immunological studies in patients suffering from sympathetic ophthalmitis.

In nine sympathetic ophthalmitis patients, the serum IgG, IgA, and IgM concentrations were found to be in the normal range, as were the proportions of active and total T-lymphocytes, the proportions of C3-binding and mouse erythrocyte-binding receptor-bearing B lymphocytes, the lymphocyte-stimulating effects of phytohemagglutinin and concanavalin A, the leukocyte migration-inhibiting effect of the purified protein derivative of tuberculin, and the suppressor activity of the peripheral blood mononuclear cells. The beta 1C (C3 component of complement) level was increased in every patient. These results indicate that sympathetic ophthalmitis may also develop without damage to the immune system.

Consequences of modified fasting in obese pediatric and adolescent patients: effect of a carbohydrate-free diet on serum proteins

Serum proteins were monitored during three studies of obese adolescents treated with protein-supplemented fasting in a clinical research center. In the first study of nine patients, small but significant decreases were noted for total serum protein, albumin, transferrin, retinol-binding protein, and complement beta 1c after 4 wk of carbohydrate-free protein-supplemented fast. In a further study of four of these same patients, the substitution of 400 glucose cal for 400 fat cal in a 5th wk of dietary study returned the total protein and retinol-binding protein concentrations to base-line levels. In a 3rd study of four patients, only complement beta 1c was significantly below base-line concentrations after 3 wk on a carbohydrate-containing protein-supplemented fast. There is an apparent metabolic effect of carbohydrate ingestion on maintaining usual concentrations of serum proteins.

Immunofluorescence study of cutaneous blood vessels of 16 patients with systemic lupus erythematosus

In 16 patients with systemic lupus erythematosus, blood vessels in the affected region of the skin were examined regarding localization of immune complexes. Deposition of IgG, IgA, IgM, IgE, beta 1C and fibrin in the vessel walls was more frequently observed in intramural and perivascular sites than in intimal sites. IgM and fibrin were usually detected in intramural sites only. In large caliber arteries of the skin the intima was the main site of deposition of immune reactants, whereas, in small-caliber vessels, including capillaries, immune reactants were more often found in intramural and perivascular sites.

The deposition of immunoglobulins and complement in osteoarthritic cartilage.

In a prospective study of 117 patients having reconstructive surgery for osteoarthritis, biopsies of hyaline articular cartilage and meniscal fibrocartilage were taken which exhibited immunofluorescence in their articular surfaces, for at least two immuno-globins (Ig) and beta 1c. These could represent immune complexes. The cases were classified into three Groups according to clinical, radiological and laboratory features, (1) non-arthritic -- 27 (3% positive); (2) secondary degenerative -- 32 (16% positive); (3) idiopathic osteoarthritic -- 89 (51% positive). The incidence of positive findings in Group III was significant at p less than 0.001 and less than 0.005 levels respectively as compared to those of Groups I and II. Our study of the disease features and their associated laboratory parameters indicated the following: positive findings correlated with an older mean age and longer disease duration. The gradings for mononuclear cell infiltration in synovial biopsies of Group III were more than two times higher than those of secondary arthritics. Group III also had an increased incidence of circulating auto-antibodies. A careful review of clinical features has not suggested a mixed population with rheumatoid disease but rather that Group III represents part of the spectrum of primary generalised osteoarthritis. These data suggest involvement of local immune mechanisms in cartilage degradation in the joints of those patients with longer term involvement.

HISTOPATHOLOGICAL ANALYSIS OF SIXTEEN SUBCUTANEOUS RHEUMATOID NODULES

An immunopathological study was carried out on subcutaneous nodules (rheumatoid nodules) biopsied from 16 patients with rheumatoid arthritis. Sixteen rheumatoid nodules were histologically classified into three stages, the 1st stage (acute inflammatory stage, 3 cases), the 2nd stage (granulomatous stage, 10 cases), and the 3rd stage (scar-formed stage, 3 cases). In necrotic or granulation tissue of the nodules in the 3 stages, immunofluorescent analysis using FITC-labeled rabbit anti-human immunoglobulins, beta 1C, and fibrinogen serum gamma-globulins and FITC-labeled aggregated human IgG gave results that were strongly positive in the 1st stage, moderately positive in the 2nd stage, and only slightly positive or almost negative in the 3rd stage. Acute or chronic thrombotic endoarteritis was observed around rheumatoid nodules in 6 out of the 16 cases. Among them, 2 cases of the 1st stage showed acute thrombotic endoarteritis with marked infiltration of neutrophils in the wall, and 2 cases of the 2nd and the 3rd stages respectively showed chronic endoarteritis with organized thrombi.

Demonstration of Dengue Antibody Complexes on the Surface of Platelets from Patients with Dengue Hemorrhagic Fever

By the direct immunofluorescent technic, dengue antigen, human immunoglobulins, and beta 1C globulin were detectable on the surfaces of platelet suspensions from 48% of patients with dengue hemorrhagic fever. The percentages of positive-staining platelets were not related to the severity of thrombocytopenia, which was marked on the day after the patient developed shock or subsidence of fever. It is suggested that an immunologic mechanism is one of the factors associated with the thrombocytopenia caused by increased platelet destruction.

PRE-ECLAMPSIA WITH THE NEPHROTIC SYNDROME

Heavy proteinuria and/or the nephrotic syndrome rarely occur late in pregnancy. We report the clinical and renal biopsy findings on 11 patients with the nephrotic syndrome occurring during pregnancy in whom light and electron microscopic findings were characteristic of pre-eclampsia. Immunofluorescent microscopy revealed deposits of IgG, IgM, IgA, beta 1C globulin, and fibrinogen, predominantly in the subendothelial position. Only two patients were primigravid; three were in their second or third pregnancy; in six, pre-eclampsia first occurred in the fourth to eighth pregnancy. Clinical abnormalities appeared first between the 23rd and 39th week of gestation. All patients had marked elevation of blood pressure and of serum uric acid levels. Of the 12 infants, eight were alive and well, including one set of twins; four were stillborn. Following delivery, clinical resolution was similar to that in less severe pre-eclampsia. The findings suggest the possible importance of the role of intravascular coagulation in the genesis of pre-eclampsia.

Pre-eclampsia with the nephrotic syndrome

Heavy proteinuria and/or the nephrotic syndrome rarely occur late in pregnancy. We report the clinical and renal biopsy findings on 11 patients with the nephrotic syndrome occurring during pregnancy in whom light and electron microscopic findings were characteristic of pre-eclampsia. Immunofluorescent microscopy revealed deposits of IgG, IgM, IgA, beta 1C globulin, and fibrinogen, predominantly in the subendothelial position. Only two patients were primigravid; three were in their second or third pregnancy; in six, pre-eclampsia first occurred in the fourth to eighth pregnancy. Clinical abnormalities appeared first between the 23rd and 39th week of gestation. All patients had marked elevation of blood pressure and of serum uric acid levels. Of the 12 infants, eight were alive and well, including one set of twins; four were stillborn. Following delivery, clinical resolution was similar to that in less severe pre-eclampsia. The findings suggest the possible importance of the role of intravascular coagulation in the genesis of pre-eclampsia.