Gap junction communication mediates transforming growth factor-beta activation and endothelial-induced mural cell differentiation

During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-beta (TGF-beta) activation. We investigated whether gap junction channels are formed between endothelial cells and recruited mesenchymal progenitors and whether intercellular communication is necessary for endothelial-induced mural cell differentiation. Mesenchymal progenitors from Cx43-/- murine embryos and Cx43+/+ littermates were cocultured with prelabeled endothelial cells. Intracellular dye injection and dual whole-cell voltage clamp revealed that endothelial cells formed gap junction channels with Cx43+/+ but not Cx43-/- progenitors. In coculture with endothelial cells, Cx43-/- progenitors did not undergo mural cell differentiation as did Cx43+/+ cells. Stable reexpression of Cx43 in Cx43-/- cells (reCx43) restored their ability to form gap junctions with endothelial cells and undergo endothelial-induced mural cell differentiation. Cocultures of endothelial cells and either Cx43+/+ or reCx43 mesenchymal cells produced activated TGF-beta; endothelial-Cx43-/- cocultures did not. However, Cx43-/- cells did produce latent TGF-beta and undergo mural cell differentiation in response to exogenous TGF-beta1. These studies indicate that gap junction communication between endothelial and mesenchymal cells mediates TGF-beta activation and subsequent mural cell differentiation.     

MCP-1 mediates TGF-beta-induced angiogenesis by stimulating vascular smooth muscle cell migration

Transforming growth factor-beta (TGF-beta) and its signaling mediators play crucial roles in vascular formation. Our previous microarray analysis identified monocyte chemoattractant protein-1 (MCP-1) as a TGF-beta target gene in endothelial cells (ECs). Here, we report that MCP-1 mediates the angiogenic effect of TGF-beta by recruiting vascular smooth muscle cells (VSMCs) and mesenchymal cells toward ECs. By using a chick chorioallantoic membrane assay, we show that TGF-beta promotes the formation of new blood vessels and this promotion is attenuated when MCP-1 activity is blocked by its neutralizing antibody. Wound healing and transwell assays established that MCP-1 functions as a chemoattractant to stimulate migration of VSMCs and mesenchymal 10T1/2 cells toward ECs. Furthermore, the conditioned media from TGF-beta-treated ECs stimulate VSMC migration, and inhibition of MCP-1 activity attenuates TGF-beta-induced VSMC migration toward ECs. Finally, we found that MCP-1 is a direct gene target of TGF-beta via Smad3/4. Taken together, our findings suggest that MCP-1 mediates TGF-beta-stimulated angiogenesis by enhancing migration of mural cells toward ECs and thus promoting the maturation of new blood vessels.     

An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes.

Using an in vitro coculture system to mimic the interactions between the cells of the vessel wall, we have previously shown that pericytes and smooth muscle cells (SMC) inhibit the growth of capillary endothelial cells (EC). We have undertaken studies to determine the mechanism of this inhibition. Using conditioned media and affinity-purified antibodies to transforming growth factor beta (TGF-beta), we now demonstrate that activated TGF-beta produced in these cocultures mediates EC growth inhibition. No inhibitory activity was detected when media conditioned by individual cultures of EC, SMC, or pericytes were examined for their effect on EC growth. In contrast, media conditioned by cocultures of EC-SMC and EC-pericytes inhibited EC proliferation to the same degree as the coculture itself. Immunoadsorption of coculture-derived conditioned media with antibodies to TGF-beta eliminated the inhibitory activity. Acid activation of serum-free media conditioned by any of the cells cultured alone yielded inhibitory activity, whereas activation of coculture conditioned media did not increase its inhibitory activity. Addition of anti-TGF-beta neutralizing antibodies to cocultures blocked the pericyte-mediated EC growth inhibition. These results indicate that latent TGF-beta is produced by these cells and it is activated by a mechanism that requires contact between the two cell types.     

骨髓基质干细胞种植体外修复内皮及对血管平滑肌细胞增生的影响

目的探讨骨髓基质干细胞(MSCs)种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔血管内皮、平滑肌和人MSCs,通过细胞共培养模拟血管内皮修复过程,用流式细胞仪分析MSCs分子表型特征,免疫荧光细胞化学法观察与内皮共培养的MSCsFlk1和vWF蛋白表达,根据下室内皮生长状态及是否接种MSCs将其分为对照组、单纯MSCs组、融合内皮组、对数内皮组和MSCs种植组。氚胸腺嘧啶脱氧核苷(3HTdR)掺入检测平滑肌细胞DNA合成,Westernblot检测平滑肌细胞中增殖细胞核抗原蛋白表达。结果分离的MSCs表达基质细胞标志CD105和CD166,不表达造血干祖细胞和内皮细胞标志CD34、Flk1、vWF;与内皮共培养5天时,vWF染色仍为阴性,但约25.71%MSCs开始表达Flk1;MSCs种植组平滑肌细胞3HTdR掺入虽高于融合内皮组,但与对数内皮组比较显著降低;MSCs种植组平滑肌细胞PCNA蛋白吸光度相对值虽高于融合内皮组,但与对数内皮组比较明显减少。结论MSCs种植能抑制平滑肌细胞增生,种植在成熟内皮中的MSCs具有微环境依赖向内皮分化的能力。     

Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium

OBJECTIVE: The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium. METHODS: To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents. RESULTS: EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment. CONCLUSION: This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.     

TGFβ is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells

New vessels form de novo (vasculogenesis) or from pre-existing vessels (angiogenesis) in a process that involves the interaction of endothelial cells (EC) and pericytes/smooth muscle cells (SMC). One basic component of this interaction is the endothelial-induced recruitment, proliferation and subsequent differentiation of pericytes and SMC. We have previously demonstrated that TGFβ induces the differentiation of C3H/10T1/2 (10T1/2) mesenchymal cells toward a SMC/pericyte lineage. The current study tests the hypothesis that TGFβ not only induces SMC differentiation but stabilizes capillary-like structures in a three-dimensional (3D) model of in vitro angiogenesis. 10T1/2 and EC in Matrigel™ were used to establish cocultures that form cord structures that are reminiscent of new capillaries in vivo. Cord formation is initiated within 2–3 h after plating and continues through 18 h after plating. In longer cocultures the cord structures disassemble and form aggregates. 10T1/2 expression of proteins associated with the SMC/pericyte lineage, such as smooth muscle α-actin (SMA) and NG2 proteoglycan, are upregulated in these 3D cocultures. Application of neutralizing reagents specific for TGFβ blocks cord formation and inhibits expression of SMA and NG2 in the 10T1/2 cells. We conclude that TGFβ mediates 10T1/2 differentiation to SMC/pericytes in the 3D cocultures and that association with differentiated mural cells is required for formation of capillary-like structures in Matrigel™. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hepatocyte growth factor mediates angiopoietin-induced smooth muscle cell recruitment

Communication between endothelial cells (ECs) and mural cells is critical in vascular maturation. Genetic studies suggest that angiopoietin/Tie2 signaling may play a role in the recruitment of pericytes or smooth muscle cells (SMCs) during vascular maturation. However, the molecular mechanism is unclear. We used microarray technology to analyze genes regulated by angiopoietin-1 (Ang1), an agonist ligand for Tie2, in endothelial cells (ECs). We observed that hepatocyte growth factor (HGF), a mediator of mural cell motility, was up-regulated by Ang1 stimulation. We confirmed this finding by Northern blot and Western blot analyses in cultured vascular endothelial cells. Furthermore, stimulation of ECs with Ang1 increased SMC migration toward endothelial cells in a coculture assay. Addition of a neutralizing anti-HGF antibody inhibited Ang1-induced SMC recruitment, indicating that the induction of SMC migration by Ang1 was caused by the increase of HGF. Interestingly, Ang2, an antagonist ligand of Tie2, inhibited Ang1-induced HGF production and Ang1-induced SMC migration. Finally, we showed that deletion of Tie2 in transgenic mouse reduced HGF production. Collectively, our data reveal a novel mechanism of Ang/Tie2 signaling in regulating vascular maturation and suggest that a delicate balance between Ang1 and Ang2 is critical in this process.     

Effects of human bone marrow stromal cell line (HFCL) on the proliferation,differentiation and apoptosis of acute myeloid leukemia cell lines U937, HL-60 and HL-60/VCR

This study investigated the effects of human bone marrow fibroblastoid stromal cell line (HFCL) on the proliferation, differentiation and chemosensitivity of acute myeloid leukemia cells (AML) in vitro coculture. By setting up coculture system of sensitive U937, HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cells in direct contact with human bone marrow fibroblastoid stromal cell line HFCL, or separated by transwell, the proliferation of AML cells cocultured with HFCL cells was inhibited, compared with AML cells alone. And NBT positive cells increased slightly. The percentage of G1 phase cells of AML cells cocultured with HFCL cells was higher than that without HFCL cells, and that of S phase cells was lower. The expression of CD11b and CD14 increased. Meanwhile HL-60 and HL-60/VCR cells treated by TPT were observed to have apoptosis characteristic morphological changes. The proportion of G0/G1 HL-60 and HL-60/VCR cells treated with TPT increased and the sub-G1 increased. The percentage of Annexin V-positive cells and apoptotic cells increased with expression of activated Caspase-3 and the reduced expression of Bcl-2. But when they were cocultured with HFCL cells, the percentage of Annexin V-positive cells and apoptotic cells decreased and sub-G1 reduced. After indirect contact with HFCL cells the expression of activated Caspase-3 decreased and the expression of Bcl-2 increased. After direct contact with HFCL cells for 96 h, the expression levels of 582 genes in HL-60 cells were up-regulated, and 1,323 genes were down-regulated at least twofold by Affymetrix GeneChip Human Genome U133 set A. The expression change in some genes, such as HL14, was confirmed by RT-PCR and northern blot. In a word, HFCL cells could inhibit the proliferation, induce the monocytic differentiation of U937, HL-60 and HL-60/VCR cells, and prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via modulation of Bcl-2 and active Caspase-3. Many genes might take part in the influence of HFCL cells on AML cells, which may give important insights into the interaction of bone marrow stromal cells and leukemic cells.     

Angioimmunoblastic T cell lymphoma associated with reversible myelofibrosis

A 56-year-old man complained of fever, anemia, thrombocytopenia and lymph node swelling. Biopsy of the lymph node demonstrated angioimmunoblastic T cell lymphoma (AITL) with the loss of normal architecture, proliferation of neoplastic T cells, small vessels mixed with eosinophils and plasma cells. Aspiration of bone marrow was dry tap, and biopsy demonstrated myelofibrosis with increased proliferation of reticulin fiber. Markedly elevated plasma levels of transforming growth factor beta1 (TGF-beta1) and soluble interleukin-2 receptor (sIL-2R) were observed, and that of platelet growth factor (PDGF) AB was slightly elevated. After chemotherapy, remission of lymphoma was achieved. The aspiration of bone marrow became possible, and the level of TGF-beta1 and PDGF AB showed normalization; thus, myelofibrosis was reversible.     

Development of a coculture system and use of confocal laser fluorescent microscopy to study human microvascular endothelial cell and mural cell interaction

In the present study, human myometrial microvascular endothelial cells (HMMEC) were cocultured with human vascular smooth muscle cells (VSMC) labeled with fluorescent dyes to examine their morphological interaction using confocal laser fluorescent microscopy. HMMEC and VSMC labeled with fluorescent green and red dyes, respectively, attached to opposite sides of polyethyleneterephthalate membranes and remained viable for up to 96 h. In defined medium, 5%+/-3% of the VSMC cytoplasmic processes and 71%+/-17% of the HMMEC processes extended completely across the 13 microm thickness of the transmembrane. However, 41%+/-21% of the VSMC projections and 10%+/-3% of the HMMEC processes that traversed the membrane made contact with the opposing cell type. In cocultures incubated with angiopoietin-1 (Ang-1), although the number of VSMC or HMMEC projections was not significantly increased, the number of VSMC extending across the membrane and making contact with HMMEC was increased (P<0.05) to 88%+/-2%. The results of the current study demonstrate that coculture of fluorescent-labeled HMMEC and VSMC on a semipermeable transmembrane coupled with confocal laser fluorescent microscopy provides an in vitro experimental model to study morphological association of microvascular endothelial cells with mural cells. We propose that this system will greatly facilitate study of remodeling of the microvasculature in various organ systems.     

Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression

The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured. VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay. Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe. Immunohistochemistry was performed to determine which types of cell produce VEGF. Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively. Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h. Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF. Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production. Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs. A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell. An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures. Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.     

Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress

E-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH(2)-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-kappaB (NF-kappaB)-promoter binding activity in ECs; inhibition of NF-kappaB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1beta and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm(2) inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.     

The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells

After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.     

紫杉醇联合骨髓基质干细胞种植体外修复内皮及对血管平滑肌增生的影响

目的探讨紫杉醇联合骨髓基质干细胞种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔主动脉内皮、平滑肌和人骨髓基质干细胞,通过细胞共培养将内皮/骨髓基质干细胞接种于下室、平滑肌细胞接种于上室模拟血管内皮修复过程,分别用3H TdR掺入和Westernblot检测紫杉醇(1,10,100nmol/L)干预20min后第10天平滑肌DNA合成和PCNA蛋白表达,用免疫荧光细胞化学法观察与紫杉醇干预内皮共培养的骨髓基质干细胞vWF和Flk1蛋白表达。结果骨髓基质干细胞种植组平滑肌细胞3H TdR掺入和PCNA蛋白光密度相对值均高于融合内皮组(n=6,P<0.05),低于对数内皮组(n=6,P<0.05)。共培养前骨髓基质干细胞不表达vWF和Flk1蛋白,与紫杉醇干预内皮共培养10天时vWF染色阴性,但部分骨髓基质干细胞开始表达Flk1蛋白。结论骨髓基质干细胞种植能部分抑制紫杉醇引起的平滑肌细胞延迟增生,与紫杉醇干预内皮共培养的骨髓基质干细胞有向内皮分化的能力。     

Mediation of the inhibitory effect of thyroid hormone on proliferation of hepatoma cells by transforming growth factor-beta

Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)alpha1 or TRbeta1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-beta neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-beta was studied to delineate the repression mechanism. TGF-beta was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-beta repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-beta neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.     

Regulation of DNA synthesis in chicken adipocyte precursor cells by insulin-like growth factors, platelet-derived growth factor and transforming growth factor-beta.

Adipose tissue growth can occur by both hypertrophy and hyperplasia. The capacity for adipocyte hyperplasia in vivo resides in a population of fibroblast-like adipocyte precursor cells but the regulation of the proliferation of these cells by growth factors has not been well characterized. This study was designed to determine the effects of the insulin-like growth factors (IGF-I and IGF-II), platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) added alone or together on the proliferation of primary adipocyte precursor cells in vitro. Adipocyte precursor cell proliferation measured by [3H]thymidine incorporation into DNA was stimulated by all of these growth factors and was particularly marked with PDGF. IGF-I or IGF-II added together with TGF-beta 1 produced a greater than additive response and the effect of PDGF was synergistic with that of IGF-I at certain concentrations. Stimulation of proliferation of some cell types by TGF-beta has been linked to the secondary production of PDGF but the evidence we have suggests that this is unlikely in chicken adipocyte precursors. DNA synthesis in response to TGF-beta 1 required only a short exposure to the peptide, and conditioned medium from chicken adipocyte precursor cells previously exposed to TGF-beta had no effect on DNA synthesis when added to fresh batches of cells. Addition of TGF-beta 1 together with PDGF produced a synergistic effect whereas an additive effect would be expected if PDGF mediated the effect of TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressive effect of Sl/Sld mouse embryo-derived fibroblast cell lines on diffusible factor-dependent proliferation of mast cells

Two modes of mast cell growth are present, one dependent on diffusible growth factors (interleukins [IL] 3 and 4) and another dependent on contact with fibroblasts. The 3T3 fibroblast cell lines derived from WCB6F1-+/+ mouse embryos supported the proliferation of cultured mast cells (CMC), whereas the 3T3 fibroblast cell lines from WCB6F1-Sl/Sld mouse embryos did not. To investigate the relationship between growth factor-dependent and fibroblast-dependent growths of mast cells, we cocultured CMC and 3T3 fibroblasts in the presence of diffusible growth factors. WCB6F1-+/+ mouse embryo-derived 3T3 cells did not affect the growth factor-dependent proliferation of CMC, but WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells significantly suppressed the proliferation. Close cell-to-cell contact was necessary for the suppression. The NWS1 fibroblast cell line was established from the spleen cells of an adult WBB6F1-+/+ mouse. Although the NWS1 cell line had no supporting effect on the proliferation of CMC in the absence of diffusible growth factors, it did not suppress the proliferation of CMC induced by the growth factors. The present result suggests that a product of mutant Sl genes may be involved in the suppressive activity of WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells.     

Bone morphogenetic protein 2 induced differentiation toward superficial epithelial cells in the gastric mucosa

Background Epithelial–mesenchymal interactions are important for maintenance of the gastrointestinal tract mucosa. Moreover, diffusible factors from the underlying mesenchyme control the proliferation and differentiation of the epithelial cells. However, the details of the associated signaling remain unknown. Methods Two novel cell lines, designated MSE1 (mouse stomach epithelium) and MSMF1 (mouse stomach myofibroblast) cells, were established from mouse glandular stomach and cocultured in three-dimensional collagen gels in vitro. Results MSE1 cells formed dramatic branching tubular structures upon coculture with MSMF1 cells. In contrast, they formed spherical cyst structures in the absence of fibroblast support or the presence of Swiss 3T3 cells. Since bone morphogenetic protein 2 (BMP2) was expressed by MSMF1 cells but not Swiss 3T3 cells, we investigated whether it induced the morphological differentiation. Addition of BMP2 to MSE1 cells induced the formation of branching tubular structures, even in the absence of MSMF1 cells. Noggin, a BMP2 antagonist, blocked the MSMF1-induced tubular branch formation by MSE1 cells. MSE1 cells were induced to express mRNA of MUC5AC, an important marker for gastric superficial epithelium in the upper part of pits, upon branching tubule formation after BMP2 addition. Coculture with MSMF1 cells or BMP2 addition induced Smad1 phosphorylation in MSE1 cells. Furthermore, BMP2 inhibited MSE1 cell proliferation in MTS assays and suppressed AKT phosphorylation. Conclusions BMP2 stimulated MSE1 cells to form branching duct-like structures and differentiate toward superficial epithelium in three-dimensional cocultures in vitro, suggesting that it may act as a morphogen and differentiation inducer in epithelial–mesenchymal interactions of gastric mucosa.