Biomedical subjects

D Danielpour

Publications and source records attributed to D Danielpour.

At least 37 records · Page 2Linked to original sources

PubMed1995

Specific and sensitive quantitation of transforming growth factor beta 3 by sandwich enzyme-linked immunosorbent assay.

Transforming growth factors beta (TGF-beta) consist of a highly homologous family of 25 kDa dimers involved in a diverse array of biological functions. Progress in understanding the biology of the third isoform (TGF-beta 3) of this family has been limited by the absence of a quantitative assay for TGF-beta 3. Here we report the development of a sensitive and specific sandwich enzyme-linked immunosorbent assay (SELISA), which is based on an anti-TGF-beta mouse monoclonal IgG as the capture antibody and chicken anti-recombinant-hTGF-beta 3 IgY as the secondary antibody. This assay can quantitate TGF-beta 3 in complex biological fluids, with a detection limit of 2 pg and no cross-reactivity or interference with as high as 1000-fold molar excesses of either TGF-beta s 1, 2 or 1.2. This TGF-beta 3 SELISA is the first reported assay for the direct and sensitive quantitation of TGF-beta 3 in complex biological fluids.

Animals↗

PubMed1995

Transforming growth factor-beta1 circulates in normal human plasma and is unchanged in advanced metastatic breast cancer.

A method has been developed to determine true plasma transforming growth factor beta (TGF-beta) levels by using the platelet alpha granule-specific marker, platelet factor 4, to correct for the TGF-beta contributed by platelets degranulated ex vivo. TGF-beta levels were measured on acid-ethanol extracts of human plasma using isoform-specific sandwich enzyme-linked immunosorbent assays. Normal human subjects had 4.1 +/- 2.0 ng/ml TGF-beta1 (range, 2.0-12.0; n = 42), <0.2 ng/ml TGF-beta2, and <0.1 ng/ml TGF-beta3 in their plasma. There were no significant changes with age or with hormonal status, but any given individual showed fluctuations of up to 3-fold in measured plasma TGF-beta levels due to unknown factors. Of 28 patients with advanced metastatic breast cancer, 2 had greatly elevated TGF-beta1 levels, while the rest were in the normal range. The presence of physiologically significant levels of TGF-beta1 in the plasmas of normal human subjects may indicate previously unsuspected endocrine roles for this peptide, while TGF-beta2 and TGF-beta3 appear to act only in a local autocrine/paracrine fashion.

Adult↗

PubMed1994

Abnormal cytokine expression in Sézary and adult T-cell leukemia cells correlates with the functional diversity between these T-cell malignancies.

The Sézary syndrome (SzS) and adult T-cell leukemia (ATL) are malignant proliferations of mature T-lymphocytes that possess distinct functions. Sézary cells function as helper cells, whereas ATL cells are usually suppressor effectors. Although phenotypically similar (CD4+/CD7-/CD8-), these functional differences between the T-cell lymphoproliferative disorders suggest different patterns of cytokine expression. We wished to delineate the cytokine mechanisms potentially underlying the diverse functional characteristics of SzS and ATL. Therefore, we analyzed the expression of interleukins (IL) 2, 4, and 5, gamma-interferon, and transforming growth factor beta 1 in the highly purified leukemic T-cells from 5 SzS and 5 ATL patients. Decreased mRNA and protein levels of IL-2, gamma-interferon, and IL-5 were detected in mitogen-stimulated ATL and SzS cells when compared to similarly cultured normal CD4+ cells. In contrast, IL-4 production was markedly up-regulated in the leukemic cells of 4/5 SzS patients as compared to ATL and normal controls. Finally, fresh ATL cells secreted higher levels of transforming growth factor beta 1 into the culture medium than the malignant T-cells from SzS patients. Collectively these results suggest that, similar to the murine CD4-expressing T-cell subsets Th1 and Th2, different cytokine profiles exist in a human population of CD4+ T-cells. Moreover, the distinct patterns of IL-4 and transforming growth factor beta 1 expression by SzS and ATL cells, respectively, are most consistent with the functional differences (i.e., helper versus suppressor activity) between these T-cell malignancies and thus may play important roles in the pathogenesis of the paraneoplastic features associated with these two leukemias.

Adult↗

PubMed1994

Development and characterization of nontumorigenic and tumorigenic epithelial cell lines from rat dorsal-lateral prostate.

We have established two new epithelial cell lines (NRP-152, NRP-154), with markedly different properties, from the dorsal-lateral prostate of Lobund/Wistar rats treated with N-methyl-N-nitrosourea and testosterone propionate. NRP-152 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic epithelial cells. They produce prostatic acid phosphatase, have functional androgen receptors, and require the combination of several growth factors in addition to serum for optimal growth. Their growth is stimulated by epidermal growth factor, insulin, dexamethasone, cholera toxin, dihydrotestosterone, and testosterone, and their growth is inhibited by transforming growth factor beta s and retinoic acid. These cells also respond to 1,25-dihydroxyvitamin D3 with an early growth stimulation followed by growth inhibition at later times. In contrast, tumorigenic NRP-154 cells lack detectable androgen receptor mRNA and have less stringent growth factor requirements for optimal growth. Growth of NRP-154 cells is stimulated by dexamethasone and insulin, inhibited by transforming growth factor beta 1, but not significantly altered by epidermal growth factor, cholera toxin, dihydrotestosterone, retinoic acid, or 1 alpha,25-dihydroxyvitamin D3. Our data suggest that the NRP-152 and NRP-154 cell lines are suitable systems for analysis of normal prostate growth and prostatic carcinogenesis.

Acid Phosphatase↗

PubMed1994

Interleukin-2 suppresses activated macrophage intracellular killing activity by inducing macrophages to secrete TGF-beta.

Phorbol myristate acetate (PMA) treatment of an EL-4 thymoma cell line (EL-4FARRAR) induced secretion of a factor that inhibited intracellular killing of Leishmania major amastigotes by activated macrophages. Analysis of the cytokines produced by EL-4 cells after PMA stimulation identified interleukin-2 (IL-2, 2500 U/ml), IL-4 (1280 U/ml), interferon-gamma (IFN-gamma; 100 U/ml), and granulocyte-macrophage colony-stimulating factor (GM-CSF; 50 U/ml). Neither tumor necrosis factor nor transforming growth factor beta (TGF-beta) was detected. Each of the cytokines present in EL-4 fluids was assessed for capacity to activate macrophages for destruction of parasites or to suppress intracellular killing. IFN-gamma and GM-CSF both activated macrophages to kill Leishmania; IL-2 and IL-4 had no activity for induction of this antimicrobial effector function. IL-2 and IL-4 were tested for their capacity to inhibit lymphokine- or IFN-gamma-induced destruction of L. major by macrophages: IL-4 was ineffective, but IL-2 markedly suppressed the activation of macrophages for intracellular killing. Addition of > or = 10 U/ml of IL-2 at the time of infection, or up to 4 h before, blocked up to 100% of the capacity of activated macrophages to kill intracellular amastigotes. Immunoaffinity treatment of EL-4 fluids with anti-IL-2 antibody resulted in > 80% reduction in suppression of intracellular killing. The suppressive effects of IL-2 were not direct, but mediated by TGF-beta. IL-2 induced resident peritoneal macrophages to secrete > 5000 pg/ml TGF-beta 1, a quantity that is > 500-fold higher than constitutive background levels (20-40 pg/ml) and is sufficient to block intracellular killing activities. This increase in secretion of TGF-beta was not dependent increases in TGF-beta 1 mRNA. Treatment of cultures with EL-4 fluids or recombinant IL-2 in the presence of antibody to TGF-beta 1 blocked the suppressive activity of both. Thus, IL-2 was the major suppressor factor in EL-4 fluids, and it acted indirectly through the induction and autocrine action of TGF-beta.

Animals↗

PubMed1994

Transforming growth factor beta 2 is the predominant isoform in the neural retina, retinal pigment epithelium-choroid and vitreous of the monkey eye.

Several techniques were utilized to assess the levels, disposition and cellular sources of isoforms 1 and 2 of transforming growth factor beta (TGF-beta) in the posterior pole of the monkey eye. Freshly dissected tissues, as well as the saline vehicles in which dissections were performed, were analysed by sandwich enzyme-linked immunosorbent assay. In all tissues TGF-beta 2 was the predominant isoform, with beta 2:beta 1 ratios of 6:1 for neural retina (as ng g-1) and 425:1 for vitreous (as pmol l-1). Retinal pigment epithelium (RPE)-Bruch's membrane-choroid complex contained approximately 10 times the amount of both TGF-beta isoforms as neural retina. For first passage cultures of monkey RPE, TGF-beta 2, but not TGF-beta 1, accumulated over time in conditioned media samples. Immunoreactivity for TGF-beta 2 was detected both in tissue sections of posterior pole, specifically in rod outer segments and RPE, and also in the first passage cultures of RPE. Antibodies to specific peptide sequences of both isoforms localized TGF-beta to the outer segments of rod photoreceptors. The apparent sequestration of TGF-beta 2 in photoreceptor outer segments, as well as the in vitro evidence for possible synthesis and release by RPE, suggest that TGF-beta 2 is an important modulator of visual function acting at the retina-RPE interface.

Animals↗

PubMed1994

Human cytomegalovirus infection induces transcription and secretion of transforming growth factor beta 1.

Human cytomegalovirus (CMV) infection can elicit a transitory, but profound, immunodepression in immunocompetent individuals. Cytopathogenic destruction of CMV-infected leukocytes alone does not seem sufficient to explain this phenomenon, which suggests that immune system mediators (cytokines) may play a role in amplifying local modifications wrought by CMV infection. We reported previously that transforming growth factor beta 1 (TGF-beta 1) stimulates CMV replication (J. Alcami, C. V. Paya, J. L. Virelizier, and S. Michelson, J. Gen. Virol. 74:269-274, 1993). Since TGF-beta 1 can have profound negative effects on cell growth and immune responses, we investigated the induction of TGF-beta 1 following CMV infection of permissive fibroblasts. TGF-beta 1 promoter was activated by immediate-early (IE) proteins in the absence of infection and transactivated at 5 and 9 h after infection. TGF-beta 1 mRNA increased during the early phase of infection, suggesting that this phenomenon is due to enhanced transcription of the TGF-beta 1 gene. A comparative study of the influence of CMV infection and IE protein expression on TGF-beta 1 promoter function in permissive cells pointed to a possible cooperative role between IE proteins and protein(s) expressed during the early phase of viral infection. Induction of TGF-beta 1 by CMV infection could modify infected cells individually, surrounding tissues, and systemic immune reactions to the advantage of virus replication by both upregulating CMV replication and downregulating host immune responses.

Cytomegalovirus Infections↗

PubMed1993

Improved sandwich enzyme-linked immunosorbent assays for transforming growth factor beta 1.

Transforming growth factor beta s (TGF-beta s), a highly homologous family of 25 kDa dimers, function as both paracrine and autocrine growth modulators which relay a broad spectrum of critical biological functions. Although sensitive bioassays for TGF-beta s have been described, their interference by other growth modulators have limited their application. We have previously described a highly sensitive (20-50 pg/ml) and specific sandwich enzyme-linked immunosorbent assay (SELISA) for TGF-beta 1, based on use of rabbit anti-TGF-beta 1 as the capture antibody. Widespread use of this assay was limited by the availability of this rather low-titer antibody. This report describes improved TGF-beta 1 SELISAs with comparable sensitivities to the above assay based on available sources of commercial monoclonal and polyclonal antibodies. These newly developed SELISAs are the most sensitive, specific, precise and convenient methods for the quantitation of TGF-beta 1 in complex biological fluids.

Animals↗

PubMed1993

Regulation of alveolar macrophage transforming growth factor-beta secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat.

In a model of pulmonary inflammation and fibrosis induced by the antineoplastic antibiotic, bleomycin, we previously demonstrated that TGF-beta was markedly elevated within 7 d of bleomycin administration. At the time of maximal TGF-beta production, TGF-beta 1 was localized by immunohistochemistry to be present almost exclusively in alveolar macrophages. In this study, we have demonstrated that alveolar macrophages stimulated by bleomycin-induced injury secrete large quantities of biologically active TGF-beta 1 when explanted into tissue culture. However, alveolar macrophages from normal saline-treated rats secrete small quantities of biologically inactive TGF-beta. In contrast, splenic macrophages secrete large quantities of inactive TGF-beta and are unaffected by the intratracheal bleomycin treatment. High doses of the corticosteroid methylprednisolone given intramuscularly before and concomitantly with bleomycin administration prevented the influx of alveolar macrophages into the lungs, diminishing both the number of macrophages present in the alveoli and the total lung content of TGF-beta. However, the rate of secretion of TGF-beta by alveolar macrophages recovered from the alveoli was unchanged after corticosteroid treatment. When activated alveolar macrophages were cultured in the presence of several concentrations of dexamethasone that completely suppressed IL-1 secretion, little effect on TGF-beta secretion was observed. The findings in this study demonstrate that during bleomycin-induced injury, alveolar macrophages not only secrete large quantities of active TGF-beta 1, but are a predominant source of the enhanced TGF-beta response seen in this model. Furthermore, the alveolar macrophage secretion of TGF-beta is not inhibited by the presence of high concentrations of corticosteroids.

Animals↗

PubMed1992

Differential function of intestinal intraepithelial lymphocyte subsets.

It has been proposed that intestinal intraepithelial lymphocytes (I-IEL) perform immune surveillance of the epithelial layer (1) and regulate mucosal humoral responses to exogenous Ag (2). To better understand the functional potential of this unique population, purified murine I-IEL were analyzed phenotypically and functionally. Initial studies determined that I-IEL could be distinguished based on several phenotypic characteristics including: TCR (TCR-alpha beta vs TCR-gamma delta); Thy-1, CD45R/B220, CD5, and CD8 (CD8 alpha alpha vs CD8 alpha beta) expression. Using anti-TCR mAb, individual I-IEL subsets were activated and examined functionally. Both TCR-alpha beta and TCR-gamma delta I-IEL were found to synthesize an array of lymphokines that included IL-2, IL-3, and IL-6 but not IL-4 or IL-5. Additionally, a number of lymphokines were detected that directly influence epithelial function (IFN-gamma, TNF-alpha, and TGF-beta 1). However, the majority of the I-IEL function was localized within the Thy-1+, CD45R/B220- I-IEL subset. In addition those TCR-alpha beta I-IEL expressing the CD8 alpha beta heterodimer were more easily activated. Thus, a subset of I-IEL have the capacity to respond to TCR-mediated stimuli. The functional activities of these cells may influence both local immune cell populations as well as epithelial differentiation.

Animals↗

PubMed1992

The 260-kDa transforming growth factor (TGF)-beta binding protein in rat glomeruli is a complex comprised of 170- and 85-kDa TGF-beta binding proteins.

In a previous study (MacKay, K., Robbins, A. R., Bruce, M. D., and Danielpour, D. (1990) J. Biol. Chem. 265, 9351-9356) we showed that rat glomeruli contain transforming growth factor (TGF)-beta 1 binding proteins with apparent molecular masses of 260, 170, and 85 kDa (Gl-260, Gl-170, Gl-85) as determined by electrophoresis under nonreducing conditions. We demonstrate here that Gl-260 is a complex of 170- and 85-kDa TGF-beta binding proteins. Under denaturing conditions the integrity of Gl-260 is maintained through the cross-linking of one monomer of the disulfide-linked TGF-beta 1 homodimer to Gl-85 and of the other monomer to the 100-kDa subunit of Gl-170. In addition, some Gl-260 complexes are maintained by direct cross-linking of Gl-85 to the 100-kDa subunit of Gl-170. One-dimensional peptide maps of Gl-85 and the 100-kDa subunit of Gl-170 indicate that they have distinctly different ligand binding domains. In contrast, peptide maps of Gl-85 and the type II receptor of normal rat kidney fibroblasts are similar. The biological responses of isolated glomeruli to TGF-beta appear to parallel those of cultured glomerular cells which are without detectable Gl-170 and Gl-260 binding proteins.

Affinity Labels↗

PubMed1992

Cyclic AMP induces transforming growth factor beta 2 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line PC-3.

The standard therapy for advanced prostate cancer is androgen ablation. Despite transitory responses, hormonally treated patients ultimately relapse with androgen-independent disease that is resistant to further hormonal manipulation and cytotoxic chemotherapy. To develop an additional approach to the treatment of advanced prostate cancer, we have been studying the signal transductions controlling the growth of human androgen-independent prostate carcinoma cell lines. We report here that elevation of intracellular cAMP markedly inhibits the growth of the hormone-refractory cell line PC-3. To examine the mechanism of cAMP action in PC-3 cells, we tested the effect of the cAMP analog dibutyryl cAMP (Bt2-cAMP) on the regulation of the potent negative growth factor transforming growth factor beta (TGF-beta). Bt2-cAMP selectively induced the secretion of TGF-beta 2 and not TGF-beta 1 by PC-3 cells. This TGF-beta 2 was shown to be bioactive by using the CCL-64 mink lung cell assay. TGF-beta 1 was not activated despite being present at 3-fold higher concentrations than TGF-beta 2. Northern analysis showed that Bt2-cAMP induced an increase in the five characteristic TGF-beta 2 transcripts and had no effect on the level of TGF-beta 1 or TGF-beta 3 transcripts. TGF-beta 2 induction was only weakly enhanced by cycloheximide and was completely inhibited by actinomycin D. These data show that Bt2-cAMP induces the expression of active TGF-beta 2 by PC-3 prostate carcinoma cells, suggesting a new approach to the treatment of prostate cancer and a new molecular mechanism of cAMP action.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Differential regulation of the expression of transforming growth factor-beta mRNAs by growth factors and retinoic acid in chicken embryo chondrocytes, myocytes, and fibroblasts.

Transforming growth factor-beta (TGF-beta) autoregulates its expression in several mammalian cell types. We now report that addition of TGF-beta s 1, 2, and 3 to primary chicken embryo cells differentially affects expression of the messenger RNAs for the different TGF-beta isoforms depending on the cell type. In cultured sternal chondrocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in the steady-state levels of the messenger RNAs for TGF-beta s 2 and 3, but does not change expression of TGF-beta 4 mRNA. In contrast, in cultured cardiac myocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in expression of TGF-beta s 3 and 4 mRNAs, but does not change expression of TGF-beta 2 mRNA. Moreover, expression of TGF-beta s 2, 3, and 4 mRNAs is not affected by addition of any of the TGF-beta s to fibroblasts. Addition of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or interleukin-1 (IL-1) to these chicken cells also has differential effects on expression of the different TGF-beta mRNAs depending on the cell type. Retinoic acid also has contrasting effects on chondrocytes and myocytes either increasing or decreasing, respectively, expression of TGF-beta s 2 and 3 mRNAs and TGF-beta 2 protein. Our results indicate a complex pattern of regulation of the different TGF-beta genes by themselves as well as by PDGF, EGF, IL-1, dexamethasone, TPA, and retinoic acid in chicken embryo cells.

Animals↗

PubMed1992

Differential modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin.

Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming growth factor-beta 1, revealed increased extracellular and decreased intracellular expression of transforming growth factor-beta 1 in retinoic acid-treated, compared to vehicle-treated, skin. Transforming growth factor-beta 1 staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming growth factor-beta 1 expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming growth factor-beta 1 protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-beta 1 and TGF-beta 2 immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin deposition, which is induced by transforming growth factor-beta, was elevated in retinoic acid-treated but not sodium lauryl sulfate-treated skin. Cultured adult human keratinocytes also expressed predominantly transforming growth factor-beta 1 protein, as measured by ELISA, and mRNA. Treatment of keratinocytes with retinoic acid resulted in a 50% induction of transforming growth factor-beta 1 protein, without any detectable change in transforming growth factor-beta 2. These data demonstrate disassociation of modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming growth factor-beta 1 expression were observed in both retinoic acid- and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid-treated skin.

Adult↗

PubMed1992

Porcine granulosa cells do not express transforming growth factor-beta 2 (TGF-beta 2) messenger ribonucleic acid: molecular basis for their inability to produce TGF-beta activity comparable to that of rat granulosa cells.

In contrast to rat granulosa cells (GC), GC of the pig and cow produce very low levels of transforming growth factor-beta (TGF-beta)-like activity in vitro. Because cultured rat GC predominantly express TGF-beta 2 messenger RNA (mRNA) and secrete high levels of the protein, we hypothesized that TGF-beta 2 mRNA expression by porcine GC might be absent or diminished, thus providing a molecular explanation(s) for their relatively low levels of TGF-beta production. We tested this hypothesis by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analysis. When analyzed by RT-PCR, porcine GC RNA from 1-3 mm follicles did not yield the expected 489 base pair (bp) TGF-beta 2 product but instead generated a smaller than anticipated 240 bp species; porcine testis RNA generated both the 240 and the anticipated 489 bp products. Sequencing these species indicated that the smaller form was not a novel TGF-beta 2 splice variant, and that the 489-bp product was porcine TGF-beta 2. This is the first reported nucleotide sequence for porcine TGF-beta 2; it is 90% and 91% identical to murine and human TGF-beta 2 sequences, respectively. Further RT-PCR analysis of porcine GC RNA resulted in the identification of bp products representing TGF-beta 1 and TGF-beta 3 mRNA. Enzyme-linked immunosorbent assay analysis of porcine GC conditioned medium confirmed the presence of TGF-beta 1 at very low levels. TGF-beta 2 was undetectable. Comparable analysis of GC from the diethylstilbestrol-treated prepubertal rat demonstrated the presence of TGF-beta 1 and TGF-beta 3 mRNA by RT-PCR and very low levels of the corresponding protein products in conditioned culture medium. Collectively, these results suggest that the inability of porcine GC to express TGF-beta 2 mRNA could explain the very low levels of TGF-beta activity secreted by these cells in vitro.

Animals↗

PubMed1992

Regulation of expression of transforming growth factor-beta 2 by transforming growth factor-beta isoforms is dependent upon cell type.

The effect of three different isoforms of transforming growth factor-beta (TGF-beta) on the expression of TGF-beta 2 mRNA was studied in several continuous tumor cell lines. As previously reported for the mouse fibroblast cell line AKR-2B, the expression of TGF-beta 2 mRNA transcripts of 5.4, 4.7, 3.7 and 3.0 kb was decreased after a 24 hr treatment with 5 ng/ml of TGF-beta 1, TGF-beta 2 or TGF-beta 3. In A549, HBL-100 and BSC-1 epithelial cell lines, five distinct TGF-beta 2 mRNA transcripts of 5.8, 5.1, 4.0, 3.8 and 2.8 kb were detected by Northern blot analysis. Treatment of these cells with TGF-beta 1, TGF-beta 2 or TGF-beta 3 for 24 hr resulted in a 2-3 fold increase in the 5.8, 4.0 and 3.8 kb transcripts, with little detectable change in abundance of the 5.1 and 2.8 kb transcripts. The effect of the TGF-beta proteins was dose (5 ng/ml) and time (3-6 hr) dependent. A similar 2-3 fold increase in the level of secreted TGF-beta 2 was observed following treatment of A549 cells with TGF-beta 1. Basal level and induced expression of TGF-beta 2 mRNA in response to TGF-beta isoforms was decreased in the presence of actinomycin D. In all cell lines studied, the expression of the 2.5 kb TGF-beta 1 mRNA was relatively unchanged or markedly increased in response to treatment with TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Retinoic acid induces secretion of latent transforming growth factor beta 1 and beta 2 in normal and human papillomavirus type 16-immortalized human keratinocytes.

Similar cellular responses are elicited by retinoic acid (RA) and transforming growth factor beta (TGF-beta). We investigated the ability of RA to modulate the production of TGF-beta in normal human keratinocytes (HKc) and HKc lines immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16). RA treatment of both normal HKc and HKc/HPV16 resulted in a 2-3-fold induction in secreted levels of latent TGF-beta. The induction in TGF-beta secretion by RA was dose dependent, with significant increases observed with RA concentrations as low as 1-10 nM, and time dependent, with maximal induction occurring about 3 days after initiation of RA exposure. In addition, RA induced intracellular levels of TGF-beta almost 5-fold. Sandwich enzyme-linked immunosorbent assays were used to specifically quantify TGF-beta 1 and TGF-beta 2 secreted by normal HKc and HKc/HPV16 cultured in the absence or presence of RA. RA increased the secreted levels of latent TGF-beta 1 and TGF-beta 2 an average of 2- and 5-fold, respectively, with no major differences in the fold induction between normal HKc and HKc/HPV16. Northern blot analysis of mRNA isolated from HKc/HPV16 demonstrated that RA treatment induced specific transcripts for TGF-beta 1 and TGF-beta 2 about 3- and 50-fold, respectively. RA treatment of HKc had no significant effect on the binding affinity of TGF-beta for its receptors or receptor number. Normal HKc and HKc/HPV16 displayed similar dose-dependent inhibition of proliferation by TGF-beta 1. These studies indicate that RA may regulate growth control in both normal HKc and HKc/HPV16 by enhancing TGF-beta 1 and TGF-beta 2 production, which, after activation at the cell surface, could inhibit cellular proliferation in an autocrine and/or paracrine manner.

Cell Division↗

PubMed1991

Transforming growth factor-beta in calf articular cartilage organ cultures: synthesis and distribution.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to play a prominent role in controlling proteoglycan synthesis and breakdown as measured following addition to organ cultures of calf articular cartilage (Morales, T. I., and Roberts, A. B., J. Biol. Chem., 263, 12,828-12,831, 1988). In this study, we compare two closely related TGF-beta isoforms, TGF-beta 1 and TGF-beta 2, both by assessing the effects of exogenous peptide as well as by analyzing the biosynthesis and total amount of these two isoforms in cartilage explants. Added exogenously, TGF-beta 1 and TGF-beta 2 induce a comparable increase in proteoglycan synthesis over basal controls with saturation at approximately 5 ng/ml. Synthesis of TGF-beta by basal calf cartilage cultures is demonstrated by (i) immunolocalization of intracellular TGF-beta, (ii) Northern blot analysis of steady-state mRNA levels, and (iii) immunoprecipitation of metabolically labeled TGF-beta from tissue extracts and conditioned culture medium. The net amount of extractable TGF-beta 1 and TGF-beta 2 in the basal cartilage cultures was assessed by a functional assay involving inhibition of proliferation of CCL-64 mink lung epithelial cells and by sandwich enzyme-linked immunosorbent assay. The predominant isoform was TGF-beta 1 (60-85%) and the total TGF-beta 1 + TGF-beta 2 was in excess of the amount required for maximal activation of proteoglycan synthesis. The level of both isoforms was maintained relatively constant between Days 2 and 7 of culture despite a sharp (approximately two to fourfold) drop in proteoglycan synthesis. This suggests that cartilage contains a large pool of TGF-beta which is not readily accessible to the chondrocyte. We propose that much of the polypeptide is sequestered in the matrix awaiting release upon demand.

Animals↗