Inhibitory Effect Of Conditioned Medium Research Articles

B7-H3 promotes colorectal cancer angiogenesis through activating the NF-\u03baB pathway to induce VEGFA expression

Tumor angiogenesis is a hallmark of cancer and is involved in the tumorigenesis of solid tumors. B7-H3, an immune checkpoint molecule, plays critical roles in proliferation, metastasis and tumorigenesis in diverse tumors; however, little is known about the biological functions and molecular mechanism underlying B7-H3 in regulating colorectal cancer (CRC) angiogenesis. In this study, we first demonstrated that the expression of B7-H3 was significantly upregulated and was positively associated with platelet endothelial cell adhesion molecule-1 (CD31) level in tissue samples from patients with CRC. In addition, a series of in vitro and in vivo experiments showed that conditioned medium from B7-H3 knockdown CRC cells significantly inhibited the migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs), whereas overexpression of B7-H3 had the opposite effect. Furthermore, B7-H3 promoted tumor angiogenesis by upregulating VEGFA expression. Recombinant VEGFA abolished the inhibitory effects of conditioned medium from shB7-H3 CRC cells on HUVEC angiogenesis, while VEGFA siRNA or a VEGFA-neutralizing antibody reversed the effects of conditioned medium from B7-H3-overexpressing CRC cells on HUVEC angiogenesis. Moreover, we verified that B7-H3 upregulated VEGFA expression and angiogenesis by activating the NF-κB pathway. Collectively, our findings identify the B7-H3/NF-κB/VEGFA axis in promoting CRC angiogenesis, which serves as a promising approach for CRC treatment.

Interleukin-4 released from human gingival fibroblasts reduces osteoclastogenesis

ObjectiveHuman gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts. DesignTwo hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at −80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay. ResultsThe hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture. ConclusionWe concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM.

TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A.

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.

Inhibition of the Differentiation of Monocyte-Derived Dendritic Cells by Human Gingival Fibroblasts

We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

Abstract 3786: Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration.

Abstract Connexin 43 (Cx43) can form both gap junction channels and halves of gap junctions known as hemichannels. Hemichannels allow communication between cells and the extracellular environment. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation through fluid flow shear stress (FFSS) as well as by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug. The opening of hemichannels is known to release small molecules important for bone formation and remodeling. Here, we explore the role of Cx43 hemichannels in osteocytes and how it affects cancer cell migratory behavior. We confirm the opening of Cx43 hemichannels in the presence of AD by measuring uptake of ethidium bromide (EtBr) in osteocytic MLO-Y4 cells. As expected, an increase in EtBr uptake was observed after AD treatment. To ensure the specificity of Cx43 hemichannels, we treated the MLO-Y4 cells with Cx43 (E2) antibody, an antibody which specifically blocks Cx43 hemichannel activity. This prevented the uptake of EtBr by the MLO-Y4 cells. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell migration, we stimulated the opening of Cx43 hemichannels on MLO-Y4 osteocytes with either AD or FFSS. Conditioned media (CM) was collected after treatment and were used to incubate with metastatic MDA-MB-231 breast cancer cells and PC3 prostate cancer cells. Cell migration was measured using wound healing and transwell migration assays. The CM collected from MLO-Y4 osteocytes treated with AD or by FFSS reduced cancer cell migration in a dose-dependent manner. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody. Treatment with this antibody significantly attenuated the inhibitory effect of the CM on the migration of MDA-MB-231 and PC3 cells. Interestingly, the inhibitory effects on cancer cell migration were not observed when we used CM collected from osteoblasts, whose hemichannels were not shown to open by AD treatment. In order to determine whether ATP released from the Cx43 hemichannels in osteocytes would have an effect on cancer cell migration, we depleted ATP from the CM collected from MLO-Y4 cells using apyrase, an ATP hydrolyzing enzyme. The addition of apyrase increased MDA-MB-231 cell migration. To test the effect of purinergic signaling on breast cancer cell migration, we treated the CM with oxidized ATP (oATP), a potent inhibitor of ATP P2X receptors. The addition of oATP attenuated the inhibitory effect of CM on MDA-MB-231 cell migration. Then we stimulated the purinergic receptors with a nonhydrolyzable ATP analogue, ATPγS, the migration of MDA-MB-231 cells decreased. Together, our results point to the role of adenosine nucleotides released from Cx43 hemichannels in tumor migration and metastasis. Citation Format: Jade Z. Zhou, Jean X. Jiang. Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2013-3786

Tong Luo Jiu Nao injection, a traditional Chinese medicinal preparation, inhibits MIP-1β expression in brain microvascular endothelial cells injured by oxygen-glucose deprivation

Ethnopharmacological relevanceTong Luo Jiu Nao injection (TLJN), a modern Chinese formula based on Traditional Chinese Medicine theory, has been used to treat ischemic stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. Aim of the studyTo investigate the protective effect of TLJN on oxygen-glucose deprivation (OGD) induced-injury of brain microvascular endothelial cells (BMECs). Materials and methodsThe model of OGD was established in the primarily cultured BMECs. TLJN was added to the OGD-injured BMECs for 6h. A series of assays were used to detect the effects of TLJN on: (i) MIP-1β content in BMECs conditioned media (CM) by ELISA; (ii) MIP-1β protein expression in BMECs by western blotting and immunocytochemistry; (iii) the expression of CCR5, receptor of MIP-1β, in BMECs by western blotting; (iv) the proliferative activity of microglial cells via the Cell Counting Kit-8 (CCK-8). ResultsOur results showed that the OGD-injured BMECs presented with large amounts of secretion and expression of MIP-1β and up-regulation of CCR5. Also, the CM of OGD-injured BMECs remarkably increased the proliferative activity of microglial cells. The TLJN-treated BMECs exhibited a reduction of MIP-1β content in BMECs-CM and a down-regulation of MIP-1β and CCR5 expression. In addition, an inhibitory effect of CM of OGD-injured plus TLJN injection-treated BMECs on microglial proliferation was also found. ConclusionTLJN reduced the expression of MIP-1β and CCR5 in OGD-injured BMECs, and the CM of OGD-injured plus TLJN injection-treated BMECs inhibited the proliferative activity of microglial cells, suggesting the therapeutic potential of TLJN on ischemic cerebral vascular disease.

The cross-talk between osteoclasts and osteoblasts in response to strontium treatment: Involvement of osteoprotegerin

BackgroundThe mechanism for the uncoupling effects of Sr on bone remains to be evaluated. Osteoblasts play important roles in osteoclastogenesis through regulating receptor activated nuclear factor kappa B (RANK) ligand (RANKL) and osteoprotegerin (OPG) expression. We hypothesize that OPG plays an important role in the cross-talk between osteoclasts and osteoblasts in response to Sr treatment. Materials and methodsMC3T3E1 cells were treated with Sr chloride (0–3mM) and conditioned media were collected at 24h after the treatment. The effect of conditioned media on osteoclastogenesis was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and bone resorption pits analysis. OPG and RANKL mRNA expressions in osteoblastic cells and protein secretion in the conditioned media were analyzed with real-time PCR and ELISA assay, respectively. The role of OPG in Sr-mediated inhibition of osteoclastogenesis was further evaluated with anti-OPG antibody in pre-osteoclastic cells. The role of OPG in Sr-mediated uncoupling effects on osteoporotic bone was evaluated by an animal study. Ovariectomized rats were oral administrated with vehicle or Sr chloride for two months supplemented with anti-IgG antibody (control) or anti-OPG antibody. The effects of OPG neutralization after Sr treatment on bone metabolism were analyzed by microCT, bone histomorphometry and biochemical analysis. ResultsThe conditioned media derived from Sr-treated osteoblastic cells exerted a dose-dependent inhibitory effect on osteoclastic differentiation and resorptive activity in pre-osteoclastic cells. OPG mRNA expression and protein secretion in osteoblastic cells were significantly increased after Sr treatment. Neutralization with anti-OPG antibody abolished the inhibitory effect of conditioned media on RANKL-induced osteoclastogenesis. The uncoupling effects of Sr treatment on trabecular bone were evidenced by greater bone volume and trabecular number, greater osteoid surface and bone formation rate, while less osteoclast surface. These effects were attenuated by the OPG neutralization by anti-OPG antibody injection. ConclusionThe evidences from the in vitro and in vivo studies suggested that OPG played an important role in the uncoupling effect of Sr on bone metabolism, possibly by acting as a cross-talk molecule between osteoclasts and osteoblasts in response to Sr treatment.

A Novel Effect of Growth Hormone on Macrophage Modulates Macrophage-Dependent Adipocyte Differentiation

The GH receptor (GHR) is expressed on macrophages. However, the precise role of GH in regulation of macrophage function is unclear. We hypothesized that soluble factors including cytokines produced by macrophages in a GH-dependent manner regulate adipogenesis. We confirmed expression and functional integrity of the GHR in the J774A.1 macrophage cells. Conditioned medium (CM) from macrophages inhibited adipogenesis in a 3T3-L1 adipogenesis assay. CM from GH-treated macrophages decreased the inhibitory effect of CM from macrophages on adipogenesis. This effect on preadipocyte differentiation was active only during the first (early) phase of adipocyte differentiation. CM from stromal vascular compartment macrophages of mice with macrophage-specific deletion of the GHR exhibited more inhibitory effect on 3T3-L1 preadipocyte differentiation compared with CM from stromal vascular compartment macrophages of control mice, indicating that intact GH action in primary macrophages also increases preadipocyte differentiation. GH did not increase IGF-1 expression in macrophages. PCR array analysis identified IL-1beta as a candidate cytokine whose expression was altered by GH in macrophages. Levels of IL-1beta mRNA and protein were significantly decreased in GH-treated J774A.1 macrophages. Nuclear factor-kappaB stimulates IL-1beta gene expression, and GH induced a significant decrease in the levels of phosphorylated nuclear factor-kappaB in macrophages. IL-1beta is a known inhibitor of adipogenesis, and these results support GH-dependent down-regulation of macrophage IL-1beta expression as one mechanism for the observed increase in adipogenesis with CM from GH-treated macrophages. We conclude that GH decreases secretion of IL-1beta by the macrophage and thus in a paracrine manner increases adipocyte differentiation. These results provide a novel mechanism for GH's actions in the control of adipogenesis.

Pseudomonas aeruginosa Inhibition of Flagellin-Activated NF-κB and Interleukin-8 by Human Airway Epithelial Cells

Pseudomonas aeruginosa-induced activation of NF-kappaB and secretion of proinflammatory cytokines by airway epithelial cells require that the bacteria express flagellin. We tested whether P. aeruginosa and human airway epithelial cells secrete factors that modulated this response. Experiments were performed with both the Calu-3 cell line and primary cultures of tracheal epithelial cells. P. aeruginosa strain PAK DeltafliC (flagellin knockout) did not activate NF-kappaB or interleukin-8 (IL-8) but inhibited flagellin-activated NF-kappaB by 40 to 50% and IL-8 secretion by 20 to 25%. PAK DeltafliC also inhibited NF-kappaB induced by IL-1beta and Toll-like receptor 2 agonist Pam3CSK4. Similar inhibitions were observed with strains PAK, PAO1, and PA14. The inhibitory factor was present in conditioned medium isolated from PAK DeltafliC or Calu-3 plus PAK DeltafliC, but it was not present in conditioned medium isolated from Calu-3 cells alone or from PAK DeltafliC that had been heat treated. Inhibition by PAK DeltafliC-conditioned medium was exerted from either the apical or the basolateral side of the epithelium, was enhanced in simple Ringer's solution over that in tissue culture medium, and did not result from altered pH or depletion of glucose. The inhibitory effect of conditioned medium was abolished by boiling and appeared from filtration studies to result from effects of a factor with a molecular mass of <3 kDa. These and further studies with isogenic mutants led to the conclusion that the NF-kappaB and IL-8 response of airway epithelial cells to P. aeruginosa results from a balance of proinflammatory effects of flagellin and antiinflammatory effects of a small (<3-kDa), heat-sensitive factor(s) that is not lipopolysaccharide, C12 homoserine lactone, alginate, CIF, or exotoxin A, S, T, U, or Y.

Host Cell Cytokines Induced by Chlamydia pneumoniae Decrease the Expression of Interstitial Collagens and Fibronectin in Fibroblasts

Chlamydia pneumoniae infection has been associated with chronic obstructive airway disease (COPD), asthma, and atherosclerosis. Inflammation and airway remodeling in asthma and COPD result in subepithelial fibrosis that is characterized by the deposition of interstitial collagens and fibronectin. The progression of atherosclerosis is also accompanied by an increased production of interstitial collagens in the intima. As shown by reverse transcription-PCR and immunoblotting, infection of human fibroblasts and smooth muscle cells by C. pneumoniae TW-183 downregulated the expression of type I and III collagen and fibronectin, whereas the level of type IV collagen remained unchanged. Conditioned medium from infected fibroblasts as well as epithelial WISH cells also reduced the expression of interstitial collagens and fibronectin in uninfected cells. In experiments using blocking antibodies, beta interferon was found to contribute to the inhibitory effects of conditioned medium collected from infected fibroblasts. In contrast, downregulation of matrix protein expression by conditioned medium from epithelial cells was caused by interleukin-1alpha, which was not secreted from fibroblasts following chlamydial infection. C. pneumoniae-mediated inhibition of collagen and fibronectin expression was diminished following transfection of fibroblasts with specific small interfering RNA targeting the transcription factor CCAAT/enhancer-binding protein beta. The downregulation of interstitial collagens and fibronectin by the Chlamydia-induced host cell cytokine response may modulate tissue remodeling processes in airway diseases. In atherosclerosis the inhibition of collagen synthesis by C. pneumoniae infection may promote plaque vulnerability, thereby increasing the risk of plaque rupture.

Anti-inflammatory activity of probiotic Bifidobacterium: Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells

To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk (BFM) which is effective against active ulcerative colitis (UC) and exacerbations of UC, and to explore the immunoregulatory mechanisms. Peripheral blood mononuclear cells (PBMNC) from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult (BbrY) or Bifidobacterium bifidum strain Yakult (BbiY) to estimate the amount of IL-10 or IL-8 secreted. Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium (CM) and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-alpha, whereas no such effect was observed with heat-killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IkappaB-zeta mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM. Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.

ATP-induced proliferation of developing retinal cells: regulation by factors released from postmitotic cells in culture

ATP is an important mitogen in the developing retina and its proliferative response decreases as chick retinal cells differentiate in culture. Both non-stimulated or ATP-induced proliferative response was abolished if cycling cells were cocultured with cells from older embryos or cultured with conditioned medium (CM) from postmitotic cells. The effect of CM was dose-dependent and reversible, as removal of CM from the cultures restored both basal and ATP-induced incorporation of [3H]-thymidine. The effect of CM was also dependent on the developmental stage of the retina used to prepare the medium. As tissues from older embryos were used, inhibition of the basal and ATP-induced proliferative response of the cells increased. Similar inhibition of ATP-induced increase in [3H]-thymidine incorporation was observed using CM from purified glial cultures. Neither ARL 67156, an ecto-ATPase inhibitor, prevented nor TGF-β1 and TGF-β2 mimicked the inhibitory effect of conditioned medium. Incubation of cells with CM or ATP for 24h completely abolished the formation of [3H]-phosphoinositides induced by ATP. These effects were blocked by the P2 receptor antagonist PPADS and were not observed with dialysed CM, suggesting that agonist-dependent desensitization of P2 receptors occured in cultures incubated with CM. However, removal of small molecules such as nucleotides by dialysis did not affect the decline in the proliferative activity induced by CM, suggesting that desensitization is not responsible for the conditioned medium-dependent cell cycle arrest of early developing retinal cells in culture. These results suggest that factors released from postmitotic cells induce the arrest of retinal cells in the mitotic state, a phenomenon that is concomitant with agonist-dependent P2 receptor desensitization.

Nitric Oxide Donor Increases Osteoprotegerin Production and Osteoclastogenesis Inhibitory Activity in Bone Marrow Stromal Cells from Ovariectomized Rats

Nitric oxide (NO) has emerged as a potent regulator useful in alleviating estrogen deficiency bone loss. Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) play important roles in regulating osteoclastogenesis. Although recent studies have reported NO donor attenuation of bone loss, the effect of NO donor on OPG and RANKL expression of osteogenic stromal cells and bone microenvironment in ovariectomized rats is not fully understood. Here, we showed that optimal NO donor treatment [2,2'-(hydroxynitrosohydrazino)bis-ethanamine; 15 microm] increased OPG, but not RANKL, levels in bone marrow stromal cells from ovariectomized rats. NO donor augmentation of OPG synthesis was transcriptionally mediated. The stimulatory action of NO donor on OPG expression appeared to be regulated by tyrosine kinase-dependent activation of Cbfa1/Runx2 binding to the OPG promoter, because cell cultures pretreated with tyrosine kinase inhibitor (herbimycin A), but not with protein kinase A inhibitor (calphostain C) or protein kinase C inhibitor [(Rp)-cAMP] significantly reduced NO-augmented Runx2 activation and OPG levels. Conditioned medium from NO donor-treated cells inhibited macrophage-colony-stimulating factor and RANKL-induced osteoclast formation of macrophage-colony-stimulating factor-dependent bone marrow macrophages. Neutralization with anti-OPG antibodies abolished the inhibitory effect of conditioned medium on osteoclastogenesis. Immunohistochemical observation also showed that 2,2'-(hydroxynitrosohydrazino)bis-ethanamine increased OPG expression of osteochondral cells located at metaphyseal endosteum and calcified cartilage of proximal femurs in ovariectomized rats. These findings suggest that NO donor can be an alternative pharmacological strategy for regulating bone resorption.

Regulation by insulin-like growth factor (IGF) binding proteins of IGF-II-stimulated glycogenesis in cultured fetal rat hepatocytes.

We previously showed that when added to fresh medium, insulin-like growth factor (IGF)-II stimulates glycogen synthesis in cultured 18-day-old fetal rat hepatocytes. In the present study, we investigated the influence of 24-h culture-conditioned medium on IGF-II- and insulin-induced glycogenesis. The stimulatory effect of IGF-II (2.9-fold) on [14C]glucose incorporation into glycogen over 3 h was dose dependently inhibited by conditioned medium, whereas that of insulin (3.2-fold) was unaffected. Western ligand and immunoblot analysis of the conditioned media revealed IGF binding protein (IGFBP)-1, IGFBP-2, and IGFBP-4, with a predominance of IGFBP-1. IGFBP-3 was not detected. Preincubation of conditioned medium with IGF-II for 4 h at 4 C restored the glycogenic effect of newly added IGF-II. Preincubation of fresh medium with recombinant IGFBP-1 or IGFBP-3 in the presence of IGF-II suppressed IGF-II stimulation of glycogen synthesis. IGFBPs alone had no effect on glycogenesis. Des(1-6)IGF-II and R6IGF-II, structural analogs of IGF-II that have weak affinity for the IGFBPs, elicited maximal stimulation, near that of IGF-II (2.8-fold and 3.1-fold vs. 3.0-fold for IGF-II), whether tested in fresh or conditioned medium. The inhibitory effect of conditioned medium on IGF-II-induced glycogenesis is therefore mediated by the IGFBPs via sequestration of IGF-II. This suggests that the IGFBPs, particularly IGFBP-1, produced by fetal rat hepatocytes in culture may play a role in regulating glycogenesis.

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85–93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Requirement for receptor‐bound urokinase in plasmin‐dependent cellular conversion of latent TGF‐β to TGF‐β

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-beta) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR+), a human prouPA cDNA (LhuPA), or a control neomycin-resistance cDNA (Lneo). When LhuPAR+ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR+ cells. The preferential activation of TGF-beta by co-cultures with the greatest plasmin-generation potential, LhuPAR+ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-beta production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR+ cells. Inclusion of neutralizing antibodies to TGF-beta abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-beta. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-beta activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR+ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-beta IgG indicated that the inhibition was due to TGF-beta. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR+ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR- cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-beta as assayed by both the BAE migration and PA assays, presumably because it interfered with cell-surface localization of LTGF-beta. Additionally, small numbers of LhuPA and LhuPAR+ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-beta. These results support the conclusion that plasmin-dependent activation LTGF-beta by LB6 cells is promoted by the surface localization of uPA by its receptor.

Inhibition of human erythroid colony-forming units by tumor necrosis factor requires beta interferon.

We have previously reported that inhibition of human CFU-erythroid (E) colony formation by tumor necrosis factor (TNF) is an indirect effect mediated by a soluble factor released from a fraction of marrow accessory cells which are predominantly stromal elements (Means, R. T., Jr., E. N. Dessypris, and S. B. Krantz. 1990. J. Clin. Invest. 86:538-541). Further studies reported here identify a mediator of this effect. The inhibitory effect of recombinant TNF on marrow CFU-E is ablated by neutralizing antibodies to human beta IFN, but not by antibodies to gamma IFN or IL-1. Anti-beta IFN also neutralizes the inhibitory effect of conditioned medium prepared from marrow cells exposed to TNF. Human beta IFN inhibits colony formation by unpurified marrow CFU-E as well as highly purified CFU-E generated from peripheral blood progenitors, and limiting dilution analysis shows that this is a direct inhibitory effect. TNF has been implicated in the pathogenesis of the anemia of chronic diseases since blood TNF levels are elevated in many patients with this syndrome, and since exposure to TNF produces a similar anemia in either humans or mice. The present study demonstrates that beta IFN is a required mediator of this inhibitory effect on erythropoiesis.

Autocrine-paracrine inhibition of growth hormone and prolactin production by GH3 cell-conditioned medium.

In previous work we have shown that perifused GH3 cells exhibit spontaneously accelerating growth hormone (GH) and prolactin (PRL) secretory rates. This behavior contrasts with GH and PRL secretion rates that are decreasing or stable over the same 3-d period in static cell culture. We now report that GH3 cells maintained in serum-supplemented medium produce an autocrine-paracrine factor(s) which inhibits GH secretion in plate culture; PRL release is frequently reduced as well. The inhibitory effect of conditioned medium on GH secretion was concentration dependent, whereas PRL release was stimulated at low and inhibited at high concentrations over the same range. Extensive dialysis of conditioned medium using membranes with a molecular weight cut-off of 12,000-14,000 did not remove GH inhibition but produced a retentate that stimulated PRL secretion. Heat-inactivation of conditioned medium did not abolish inhibition of GH release but did remove the PRL-stimulatory effect. IGF-I added to fresh culture medium did not reproduce the GH-inhibitory effects of conditioned medium. We conclude that GH3 cell secretory behavior in perifusion and plate culture systems may be partially explained by the production of an autocrine-paracrine factor: its accumulation in plate culture inhibits GH and PRL secretion whereas its removal, by perifusing medium, allows GH and PRL secretion to accelerate.

Thrombin-mediated mitogenesis: the role of secreted protease nexin.

AbstractProtease nexin (PN) is a cell‐secreted protein that links to thrombin (Th) and certain other serine proteases. PN mediates the binding, internalization, and degradation of these proteases by cells (Baker et al., 1980; Low et al., 1981). Here we show that binding of Th‐PN complexes to human foreskin fibroblasts (HF cells) accounted for 90% of the specific cellular Th binding at certain mitogenic doses of the protease. However, cell‐associated Th‐PN complexes were likely to be inactive mitogenically because heparin (170 units/ml) inhibited cellular binding of 125‐Th‐PN by about 95% (a reduction from 1.3 × 105 to 6 × 103 125I‐Th‐PN complexes per cell) but did not influence Th‐mediated mitogenic stimulation. In experiments with mouse embryo cells, heparin also markedly decreased cellular binding of 125I‐Th‐PN without changing the mitogenic response to Th. The lack of mitogenic activity of cell‐associated Th‐PN complexes suggested that PN might inhibit the mitogenically essential proteolytic activity of Th. This possibility is supported by the following findings. First, amounts of serum‐free conditioned culture medium that contained enough PN to complex a large fraction of added Th inhibited the clotting activity of Th. Second, heparin increased the formation of 125I‐Th‐PN complexes and also increased this inhibitory effect of conditioned medium. We conclude that PN acts as a negative modulator of thrombin mitogenic activity.It is shown that like other fibroblastic cells HF cells bound free 125I‐Th specifically (although with relatively low affinity, Kass &lt; 108 M‐1). Specific binding of free 125I‐Th to HF cells increased fourfold in the presence of heparin (50 IU/ml).