Autocrine control of collagenase synthesis by synovial fibroblasts

Fibroblasts respond to exogenous stimuli, such as Interleukin 1, phorbol esters, or crystals of monosodium urate monohydrate, by synthesizing and secreting large quantities of collagenase. Here we show that addition of exogenous stimuli results in the production of an autologous protein that is, itself, capable of inducing collagenase. This autocrine has been partially purified. Activity resides in a protein(s) with a pl of 5 or 8 and with Mr of approximately 15K. Conversely, conditioned medium taken from unstimulated cultures contains an inhibitor of collagenase synthesis. This protein, which has a Mr approximately 20-25k by HPLC gel filtration antagonizes collagenase synthesis induced by phorbol esters, exogenous parallel 1, and the autologous inducer. We conclude that synovial fibroblasts regulate collagenase synthesis via an autocrine mechanism that includes the synthesis of both an inducer and inhibitor. Both proteins are active at nanomolar amounts and may function as polypeptide hormones in regulating collagenase synthesis and, hence, connective tissue remodeling.     

Autocrine motility factor/phosphoglucose isomerase regulates ER stress and cell death through control of ER calcium release

Autocrine motility factor/ phosphoglucose isomerase (AMF/PGI) promotes cell survival by the pAkt survival pathway. Its receptor, gp78/AMFR, is an E3 ubiquitin ligase implicated in endoplasmic reticulum (ER)-associated protein degradation. We demonstrate here that AMF/PGI also protects against thapsigargin (TG)- and tunicamycin (TUN)-induced ER stress and apoptosis. AMF/PGI protection against the ER stress response is receptor mediated as it is not observed in gp78/AMFR-knockdown HEK293 cells. However, AMF/PGI protection against the ER stress response by TG and TUN was mediated only partially through PI3K/Akt activation. AMF/PGI reduction of the elevation of cytosolic calcium in response to either TG or inositol 1,4,5-trisphosphate receptor activation with ATP was gp78/AMFR-dependent, independent of mitochondrial depolarization and not associated with changes in ER calcium content. These results implicate regulation of ER calcium release in AMF/PGI protection against ER stress and apoptosis. Indeed, sequestration of cytosolic calcium with BAPTA-AM limited the ER stress response. Importantly, elevation of cytosolic calcium upon treatment with the calcium ionophore ionomycin, while not inducing an ER stress response, did prevent AMF/PGI protection against ER stress. By regulating ER calcium release, AMF/PGI interaction with gp78/AMFR therefore protects against ER stress identifying novel roles for these cancer-associated proteins in promoting tumor cell survival.     

TGF-beta autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells.

Transforming growth factor beta (TGF) is a well-known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF-beta autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF-beta, thus decreasing the concentration of active TGF-beta to which the cells are exposed. This event is mediated by the TPA-induced alteration of the uPA/PAI serine-protease system. Complete removal of TGF-beta, mediated by the ectopic expression of a soluble type II TGF-beta receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF-beta concentration, to a range of 0.14-0.20 x 10(-2) ng/ml (which is similar to that measured in TPA-treated cells), mimics TPA-induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF-beta; furthermore, they show that a 'critical' concentration of TGF-beta is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF-beta autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF-beta is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF-beta independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF-beta in the regulation of both the transformed and differentiated phenotype.     

TGF-beta is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) inhibited secretion of human hepatocyte growth factor (hHGF), which is also known as scatter factor or fibroblast-derived tumor cytotoxic factor, by MRC-5 cells. The effect was detectable at as little as 10 pg/ml and was more potent than that of dexamethasone. Complete inhibition was observed after 12 h in the presence of 5 ng/ml of TGF-beta 1. Phorbol 12-myristate 13-acetate-induced secretion of hHGF from human skin fibroblasts was also suppressed by TGF-beta 1. TGF-beta 2 inhibited hHGF secretion by MRC-5 cells to the same extent as TGF-beta 1, but other growth factors such as epidermal growth factor and acidic and basic fibroblast growth factors had only a slight or null inhibitory effect.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

Lysophospholipid regulates release and activation of latent TGF-beta1 from chondrocyte extracellular matrix

Transforming growth factor beta-1 (TGF-beta1) is released from the extracellular matrix of rat growth plate chondrocytes and activated by stromelysin-1 (matrix metalloproteinase 3, MMP-3), an enzyme that is stored in matrix vesicles. MMP-3 is released from these extracellular organelles by the direct action of 1alpha,25(OH)2D3 via activation of phospholipase A2 (PLA2), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1alpha,25(OH)2D3 is greater in matrix vesicles from growth zone chondrocyte cultures and PLA2 activity is higher in the growth zone in vivo, suggesting that it may depend on chondrocyte maturation state in the endochondral lineage. Previous studies have shown that latent TGF-beta1 can be activated by mild detergents in vitro, suggesting that lysophospholipids may act in vivo in a similar manner. To test this hypothesis, we determined if rat costochondral growth plate cartilage cells produce lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in a maturation state-dependent manner and if LPC or LPE could release and activate latent TGF-beta1 from the extracellular matrix produced by these cells. Rat growth plate chondrocytes produced both lysophospholipids, with growth zone cells producing higher levels of LPE via PLA1, and resting zone cells producing higher levels of LPC via PLA2. LPC and LPE directly increased activation of recombinant human latent TGF-beta1 in a biphasic manner with a peak at 2 microg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-beta1. Latent TGF-beta1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [3H]-thymidine incorporation of resting zone cells, indicating the TGF-beta1 released was biologically active. LPC and LPE also released TGF-beta1 in a dose- and time-dependent manner when incubated with cell-free extracellular matrices produced by the cells. These results indicate that LPC and LPE have important roles as regulators of rat growth plate chondrocytes by directly and indirectly activating TGF-beta1 stored in the extracellular matrix.     

Autocrine and paracrine regulation of lymphocyte CB2 receptor expression by TGF-beta.

The marijuana-derived cannabinoid Delta(9)-tetrahydrocannabinol (THC) has been shown to be immunosuppressive. We report that THC induces the immunosuppressive cytokine TGF-beta by human peripheral blood lymphocytes (PBL). The ability of THC to stimulate TGF-beta production was blocked by the CB2 receptor specific antagonist SR144528 but not by the CB1 specific antagonist AM251. Furthermore, our data suggest that TGF-beta actively regulates lymphocyte CB2 receptor expression in an autocrine and paracrine manner. Whereas the addition of recombinant TGF-beta to PBL cultures downregulated CB2 receptor expression, anti-TGF-beta antibody treatment increased CB2 receptor expression. We conclude that one mechanism by which THC contributes to immune suppression is by stimulating an enhanced production of lymphocyte TGF-beta.     

Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro

Rheumatoid arthritis (RA), and not osteoarthritis (OA) synovial cells proliferate in serum-free medium, a finding that suggests that, in vitro, RA synovial cells may be stimulated to grow by the continuous autocrine production of at least one polypeptide growth factor. Adding monoclonal antibody 1D11.16, or rabbit polyclonal anti-tumor growth factor beta (anti-TGF-beta) antibodies (both neutralizing antibodies to TGF-beta 1 and TGF-beta 2) to RA synovial cells, in culture, caused a significant reduction in cell growth, an effect not seen when other growth factor antibodies (platelet-derived growth factor [PDGF], epidermal growth factor [EGF], or EGF receptor) were added to the culture medium. Taken together, these data are consistent with the concept that RA synovial cell growth in vitro is driven endogenous TGF-beta. Moreover, when EGF was added to the culture medium, this caused the numbers of RA, and not OA, synovial cells to increase significantly. This finding suggests that RA synovial cells are in G1 phase of the cell cycle; an effect that could be mediated by endogenous TGF-beta.     

Glutathione regulates transforming growth factor-beta-stimulated collagen production in fibroblasts

Transforming growth factor-beta (TGF-beta) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-beta fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGF beta decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-beta-stimulated collagen production, whereas a decrease in intracellular GSH content with L-buthionine-S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-beta-stimulated collagen production. These results suggest that GSH depletion induced by TGF-beta may mediate TGF-beta-stimulated collagen production. In addition, we showed that TGF-beta stimulated superoxide production and increased release of H2O2 from the cells, whereas GSH ester decreased basal and TGF-beta + glucose oxidase-stimulated H2O2 release. H2O2, exogenously added or continuously generated by glucose oxidase, enhanced TGF-beta-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-beta-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-beta-stimulated collagen production and that the effect of GSH depletion on TGF-beta-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling.     

Autocrine transforming growth factor alpha regulates cell adhesion by multiple signaling via specific phosphorylation sites of p70S6 kinase in colon cancer cells

Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.     

Autocrine growth and progression of murine X-ray-induced T cell lymphomas.

Primary tumors of X-ray-induced murine T cell lymphomas comprise autocrine, growth factor-dependent cells. We have grown cell lines from primary X-ray-induced thymic lymphomas (PXTLs) under conditions which minimize the progression of the cells from factor dependence to factor independence. All (22) PXTL lines grown secrete a growth factor which supports their own growth and which we will call lymphoma growth factor LGF. LGF-dependent cells are non-tumorigenic or poorly tumorigenic, do not clone in soft agar, have no detectable rearrangements in the c-myc or Pim-1 region and possess near diploid or pseudodiploid karyotypes without evidence for trisomy of chromosomes nos. 15 or 17. PXTL-secreted LGF has no interleukin 1, 2, or 3 activity nor do LGF-secreting cells synthesize detectable IL-1, -2, or -3 mRNA. LGF contains no detectable interferon or GM-CSF activity in specific bioassays. Purified EGF, TGF beta, and interleukin preparations are inactive on LGF-dependent PXTL cells. Thus LGF appears to be a new growth factor that is required for the proliferation of non-progressed T lymphoma cells. Upon progression PXTL cells become growth factor independent, are highly tumorigenic in vivo, clone in soft agar, and assume a near triploid karyotype containing numerous chromosomal aberrations. Thus in X-ray-induced lymphomagenesis an autocrine, LGF-dependent phase precedes the progressed phase characterized by rearrangements in the myc and/or Pim-1 regions as well as by many chromosomal aberrations visible in the karyotype.     

Surfactant protein A enhances apoptotic cell uptake and TGF-beta1 release by inflammatory alveolar macrophages

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-beta1 (TGF-beta1) release from both AM populations. Inflammatory AMs release twofold more TGF-beta1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-beta1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-beta1 release, and modulates the amount of TGF-beta1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.     

Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta

Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-beta binding domains. Here we show that CTGF directly binds BMP4 and TGF-beta 1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-beta 1. These results show that CTGF inhibits BMP and activates TGF-beta signals by direct binding in the extracellular space.     

Properties of a cell growth inhibitor produced by mouse embryo fibroblasts

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G-75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L-[35S] methionine-labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4-5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.     

Autocrine/paracrine actions of growth hormone in human melanoma cell lines

Melanoma is the most aggressive skin cancer. Its aggressiveness is most commonly attributed to ERK pathway mutations leading to constitutive signaling. Though initial tumor regression results from targeting this pathway, resistance often emerges. Interestingly, interrogation of the NCI-60 database indicates high growth hormone receptor (GHR) expression in melanoma cell lines. To further characterize melanoma, we tested responsiveness to human growth hormone (GH). GH treatment resulted in GHR signaling and increased invasion and migration, which was inhibited by a GHR monoclonal antibody (mAb) antagonist in WM35, SK-MEL 5, SK-MEL 28 and SK-MEL 119 cell lines. We also detected GH in the conditioned medium (CM) of human melanoma cell lines. GHR, JAK2 and STAT5 were basally phosphorylated in these cell lines, consistent with autocrine/paracrine GH production. Together, our results suggest that melanomas are enriched in GHR and produce GH that acts in an autocrine/paracrine manner. We suggest that GHR may constitute a therapeutic target in melanoma.