Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.     

Modulation of cultured chicken growth plate chondrocytes by transforming growth factor-beta 1 and basic fibroblast growth factor.

Expression of several cellular and matrix proteins which increase significantly during the maturation of growth plate cartilage has been shown to be affected by various endocrine and autocrine factors. In the studies reported here, transforming growth factor-beta (TGF-beta 1) and basic fibroblast growth factor (bFGF) were administered to primary cultures of avian growth plate chondrocytes at pre- or post-confluent stages to study the interplay that occurs between these factors in modulating chondrocytic phenotype. Added continuously to pre-confluent chondrocytes, TGF-beta 1 stimulated the cells to produce abundant extracellular matrix and multilayered cell growth; cell morphology was altered to a more spherical configuration. These effects were generally mimicked by bFGF, but cell shape was not affected. Administered together with TGF-beta 1, bFGF caused additive stimulation of protein synthesis, and alkaline phosphatase (AP) activity was markedly, but transiently enhanced. During this pre-confluent stage, TGF-beta 1 also increased fibronectin secretion into the culture medium. Added to post-confluent cells, TGF-beta 1 alone caused a dosage-dependent suppression of AP activity, but bFGF alone did not. Under these conditions, TGF-beta 1 and bFGF had little effect on general protein synthesis, but TGF-beta 1 alone caused large, dosage-dependent increases in synthesis of fibronectin, and to some extent type II and X collagens. Given together with bFGF, TGF-beta 1 synergistically increased secretion of fibronectin. These findings reveal that regulation of phenotypic expression in maturing growth plate chondrocytes involves complex interactions between growth factors that are determined by timing, level, continuity, and length of exposure.     

TGF-beta induced G(1) cell cycle arrest requires the activity of the proteasome pathway. Transforming growth factor

Transforming growth factor-beta (TGF-beta) induces a potent G(1)/S-phase cell cycle arrest of epithelial cells by inhibiting the activities of cyclin D- and cyclin E-associated kinase complexes. Downregulation of the kinase activities is mediated by induction of cyclin dependent kinase (CDK) inhibitor p15(Ink4b) which blocks CDK4 and CDK6 kinases and leads to binding of p27(Kip1) to CDK2-cyclin E complex. Levels of several of these factors are controlled by the ubiquitin-proteasome pathway. We demonstrate here that proteasomal inhibitors release the cells from TGF-beta imposed G(1)-phase arrest and instigate the entry of the cells into S-phase. Proteasomal inhibitors are shown to specifically increase the activity of the cyclin D-kinase complex by increasing the levels of p27(Kip1) and cyclin D and by maintaining CDK4/6 protein levels leading to phosphorylation of the retinoblastoma protein without increasing cyclin E-associated kinase activity. The results indicate caution in the potential therapeutic use of the proteasome inhibitors due to unscheduled initiation of DNA replication in the presence of a physiological growth inhibitor.     

Effects of transforming growth factor-beta 1 and fibronectin on SPARC expression in cultures of human periodontal ligament cells

Transforming growth factor-beta(1) (TGF-beta(1)) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-beta(1) in cultures of human periodontal ligament cells (HPL cells). TGF-beta(1) increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-beta(1) also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-beta(1). HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-alpha5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-beta(1). This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-beta(1) in HPL cells.     

Role of tumor necrosis factor-alpha and interferon-gamma in Helicobacter pylori infection

Immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contributions of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using TNF-alpha geneknockout (TNF-alpha(-/-)) mice and IFN-gamma gene-knockout (IFN-gamma(-/-)) mice. We first examined the colonizing ability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild-type and knockout mice. The number of H. pylori colonized in the stomach of IFN-gamma(-/-) and TNF-alpha(-/-) mice was higher than that of wild-type mice. These findings suggest that TNF-alpha and IFN-gamma may play a protective role in H. pylori infection. Furthermore, we examined the contribution of TNF-alpha and IFN-gamma to gastric inflammation. The CPY2052-infected TNF-alpha(-/-) mice showed a moderate infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium as did wild-type mice, whereas the CPY2052-infected IFN-gamma(-/-) mice showed no inflammatory findings even 6 months after infection. These results demonstrate that IFN-gamma may play an important role in gastric inflammation induced by H. pylori infection, whereas TNF-alpha may not participate in the development of inflammatory response.     

Transforming growth factor-alpha attenuates the acquisition of aromatase activity by cultured rat granulosa cells

The effect of transforming growth factor-alpha (TGF alpha) on granulosa cell differentiation, as assessed by the acquisition of aromatase activity, was evaluated in vitro by using a primary culture of rat granulosa cells. Harvested from immature, diethylstilbestrol-treated rats, granulosa cells were cultured under serum-free conditions for 72 hr in the presence of saturating concentrations (10(-7)M) of aromatase substrate androstenedione with or without the specific experimental agents. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen was negligible, remaining unaffected by treatment with TGF alpha (10 ng/ml) by itself. Whereas treatment with follicle-stimulating hormone (FSH) resulted in a substantial increase in the extent of aromatization, concurrent treatment with TGF alpha (10 ng/ml) resulted in significant (P less than 0.05), yet reversible inhibition (78 +/- 5.6%) of FSH action. Significantly, this effect of TGF alpha could not be accounted for by a decrease in cellular viability or plating efficiency nor by a decrease in the number of cells or their DNA content. Although independent of the FSH dose employed, the TGF alpha effect proved dose- and time-dependent, with an apparent median inhibitory dose (EC50) of 0.33 +/- 0.04 ng/ml, and a minimal time requirement of 48 hr. Capable of substantial inhibition of the forskolin-stimulated accumulation of extracellular adenosine 3', 5' cyclic monophosphate (cAMP) and estrogen, TGF alpha had a measurable albeit limited effect on N6, 2-'O-Dibutyryladenosine 3':5'-cyclic monophosphate-supported estrogen production. Relative potency comparison revealed epidermal growth factor (EGF; EC50 = 0.24 +/- 0.03 ng/ml) and TGF alpha to be virtually equipotent as regards the attenuation of FSH-stimulated estrogen biosynthesis. Taken together, our findings indicate that TGF alpha, like EGF, acting at subnanomolar concentrations, is capable of attenuating the FSH-stimulated (but not basal) accumulation of estrogen. This effect of TGF alpha proved time- and dose-dependent, involving virtually complete neutralization of FSH action at site(s) both proximal and distal to cAMP generation. As such, these findings provide yet another example of the remarkable qualitative and quantitative similarities between EGF and TGF alpha, thereby reaffirming the prospect that ligands of the EGF/TGF alpha receptor may play a modulatory role in the course of granulosa cell ontogeny.     

Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.     

Transforming growth factor-beta1 signaling participates in the physiological and pathological regulation of mouse inner ear development by all-trans retinoic acid

BACKGROUND: Retinoic acid (RA) is a vitamin A derivative that participates in patterning and regulation of inner ear development. Either excess RA or RA deficiency during a critical stage of inner ear development can produce teratogenic effects. Previous studies have shown that in utero exposure of the developing mouse inner ear to a high dose of all-trans RA (atRA) results in severe malformations of the inner ear that are associated with diminished levels of endogenous transforming growth factor-beta1 (TGF-beta(1)) protein. METHODS: In this study, the effects of a teratogenic level of atRA on levels and patterns of expression of TGFbeta receptor II (TGFbetaRII) and Smad2, a downstream component of the TGFbeta signal transduction pathway, are investigated in the developing mouse inner ear. The expression pattern of endogenous RA receptor alpha (RARalpha) and the ability of an RARalpha(1)-specific antisense oligonucleotide (AS) to modulate otic capsule chondrogenesis are demonstrated in the inner ear and in culture. RESULTS: Endogenous TGFbetaRII and Smad2 are downregulated in the inner ear following in utero atRA treatment. In addition, a reduction in endogenous TGFbeta(1) and a marked suppression of chondrogenesis occur in RARalpha(1) AS-treated cultures in comparison to untreated or oligonucleotide-treated control cultures. This chondrogenic suppression can be partially overcome by supplementation of RARalpha(1) AS-treated cultures with exogenous TGFbeta(1) protein. CONCLUSIONS: Our findings support a role for TGFbeta in the physiological and pathological effects of RA on inner ear development.     

Ghrelin attenuates plasminogen activator inhibitor-1 production induced by tumor necrosis factor-alpha in HepG2 cells via NF-kappaB pathway

Plasminogen activator inhibitor type 1 (PAI-1), produced partly from liver is a risk factor for macrovascular and microvascular complications of diabetes. Ghrelin, a recently described orexigenic peptide hormone, attenuates PAI-1 induced by TNF-alpha in the human hepatoma cell line (HepG2). Exposure to TNF-alpha (1 ng/ml) for 24h caused a significant increase in PAI-1 mRNA expression and protein secretion, as evaluated by RT-PCR and ELISA, but pretreatment with ghrelin (1-100 ng/ml) inhibited both basal and TNF-alpha-induced PAI-1 release in a dose and time-dependent manner in HepG2. PDTC, selective NF-kappaB inhibitor, had no additive inhibitory effects with ghrelin. The results indicate that ghrelin inhibits both basal and TNF-alpha-induced PAI-1 production via NF-kappaB pathway in HepG2 cells, and suggest that the peptide plays a therapeutic role in atherosclerosis, especially in obese patients with insulin resistance, in whom ghrelin levels were reduced.     

Expression of recombinant human tumor necrosis factor-α in a baculovirus expression system

A cDNA fragment coding human tumor necrosis factor-alpha (TNF-α) was inserted into the vector pSXIVVI⁺X3 with the control of Syn XIV promoter. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). Cells infected with recombinant virus synthesized TNF-α protein at a level of about 38% of total cellular protein. TNF-α activity in infected cells was measured by L929 cytotoxic assay, the highest expression level, 1.5 × 10⁴ U/10⁶ cells, was obtained at 76 h after infection. Western blot analysis of protein extracts from infected larvae showed that the virus-mediated TNF-α had immunoreactivity.     

Modulation of IL-4-induced human IgE production in vitro by IFN-gamma and IL-5: the role of soluble CD23 (s-CD23)

IL-4 specifically induced IgE production by peripheral blood lymphocytes or by tonsil or spleen cells from healthy donors. IL-4-induced IgE synthesis was dependent on CD4+ T cells and monocytes and was blocked by IFN-gamma, IFN-alpha, and prostaglandin E-2 (PGE-2). These substances also inhibited IL-4-induced CD23 expression and subsequent release of soluble CD23 (s-CD23). In addition, IgE production was blocked by F(ab')2 fragments of an mAb against CD23. In contrast, IL-5 enhanced IL-4-induced IgE production, provided IL-4 was added at nonsaturating concentrations. This increase in IgE production correlated quantitatively with an enhanced release of s-CD23. Collectively, these results indicate that there is a correlation between s-CD23 release and IgE production. However, s-CD23 fractionated from supernatants of the lymphoblastoid cell line RPMI-8866 was ineffective in inducing IgE production in the absence of IL-4, but acted synergistically with suboptimal concentrations of IL-4. In addition, it is demonstrated that alloreactive T-cell clones produced varying concentrations of IL-4, IL-2, or IFN-gamma upon stimulation. Only supernatants of 2/4 of these T-cell clones induced a low degree of IgE synthesis, but in the presence of anti-IFN-gamma antibodies, all four supernatants induced a strong induction of IgE production. This IgE synthesis was blocked specifically by anti-IL-4 antibodies, indicating that IL-4 is the sole inducer of IgE synthesis. Our findings demonstrate that IL-4-induced IgE production involves complex interactions of T cells, B cells, and monocytes and is positively modulated by IL-5 and s-CD23 but down-regulated by IFN-gamma, IFN-alpha, and PGE-2, respectively.     

Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system

Transforming growth factor-beta1 (TGF-beta1) is secreted by most cells as a high molecular weight latent complex, which consists of latent TGF-beta1 disulfide bonded to latent TGF-beta1-binding protein (LTBP). Current recombinant expression systems yield less than 1-2 mg of the mature TGF-beta1 per liter of cell culture medium. In an effort to produce large quantities of the recombinant cytokine for structural studies, we have constructed a mammalian expression system based on a modified pcDNA3.1(+) vector with a glutamine synthetase gene inserted for gene amplification. The leader peptide of TGF-beta1 was replaced with that of rat serum albumin, and an eight-histidine tag was inserted immediately after the leader sequence to facilitate protein purification. In addition, Cys 33 of TGF-beta1, which forms a disulfide bond with LTBP, was replaced by a serine residue. The resulting expression construct produced a stable clone expressing 30 mg of mature TGF-beta1 per liter of spent medium. Purified TGF-beta1 bound with high affinity to its type II receptor with a solution dissociation constant of approximately 70 nM, and was fully active in both a Mv1Lu cell growth inhibition assay and in a PAI-1 luciferase reporter assay. Owing to similarities in the synthesis, secretion, and structure of TGF-beta family members, this recombinant expression system may also be applied to the overexpression of other TGF-beta isomers and even to members of the TGF-beta superfamily to facilitate their preparation.     

Induction of interleukin-1 beta production in human dermal fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha. Involvement of protein kinase-dependent and adenylate cyclase-dependent regulatory pathways.

It has previously been demonstrated that interleukin-1 (IL-1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL-1 beta production by cultured human dermal fibroblasts. We have shown that IL-1 beta is constitutively expressed as a cell-associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) exerted a dose-dependent stimulation on the production of the cell-associated IL-1 beta, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL-1 beta production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 microgram/ml) of PGE2. In contrast, higher concentrations (0.1 and 1 micrograms/ml) of PGE2, as well as exogenous dibutyryl-cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL-1 alpha and TNF-alpha. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL-1 beta expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2-induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL-1 alpha and TNF-alpha, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL-1 beta induction by the two cytokines studied.     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on enterocyte shedding of syndecan-1 and associated decreases in internalization of Listeria monocytogenes and Staphylococcus aureus

Syndecan-1 is a heparan sulfate proteoglycan expressed on epithelia, and its ectodomain can be shed into the extracellular milieu, affecting a variety of cellular functions. Using two bacteria known to react with heparan sulfate, Listeria monocytogenes and Staphylococcus aureus, experiments were designed to clarify the effect of syndecan-1 shedding on bacterial internalization by human HT-29 enterocytes. Mature enterocytes were incubated with tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma for 16h prior to addition of bacteria. These cytokines acted synergistically to decrease syndecan-1 expression, assessed by visual observations of syndecan-1 expression on enterocytes using immunohistochemistry and a monoclonal antibody to the syndecan-1 core protein, by quantifying this fluorescent intensity, and by quantifying the concentration of shed syndecan-1 using an enzyme-linked immunoabsorbent assay. Neither IFN-gamma nor TNF-alpha alone had a noticeable effect on L. monocytogenes internalization, but a mixture of both cytokines resulted in decreased (P<0.01) internalization. Enterocyte preincubation with TNF-alpha alone, and with both cytokines, was associated with decreased S. aureus internalization, at P<0.05 and P<0.01, respectively. Thus, TNF-alpha and IFN-gamma acted synergistically to shed syndecan-1 ectodomains from HT-29 enterocytes, and shedding was associated with decreased internalization of two pathogenic bacteria, suggesting that syndecan-1 shedding may modulate the pathogenesis of specific microbes.