Transforming Growth Factor-β1 Differentially Regulates Proliferation, Morphology, and Extracellular Matrix Expression by Three Neural Crest-Derived Neuroblastoma Cell Lines

We reported previously (S. L. Rogers, P. J. Gegick, S. M. Alexander, and P. G. McGuire, Dev. Biol. 151, 191-203, 1992) that transforming growth factor-β1 (TGFβ1) inhibited proliferation, up-regulated fibronectin synthesis, and suppressed melanogenesis in a population of quail neural crest cells in vitro. Here, we report that cell lines derived from the parent SK-N-SH neuroblastoma line (R. A. Ross, B. A. Spengler, and J. L. Biedler, J. Natl. Cancer Inst. 71, 741-747, 1983) respond differentially to TGFβ1, and their responses provide further insights into the actions of this growth factor on neural crest subpopulations. The SH-EP cell line exhibits primarily nonneuronal traits and responded to TGFβ1 with increased thymidine uptake after 6 days of culture, increased expression of fibronectin mRNA and protein, and decreased laminin synthesis. Many SH-EP cells also acquired a dramatically elongated morphology, reminiscent of Schwann cells in culture. Thymidine uptake by the neuronal SY5Y cell line was not substantially altered. Neither fibronectin mRNA nor protein was detectable in either TGFβ1-treated or untreated cultures, although laminin synthesis was upregulated by the growth factor. In TGFβ1-treated cultures of the intermediate SH-IN cell line, which has been reported to display both neuronal and nonneuronal characteristics, there was marked flattening of many cells, a steady decrease in thymidine uptake, and increased expression of both fibronectin and laminin. The observed responses of SH-IN cells mimic those observed in primary neural crest cultures and appear to represent similar differentiation toward a mesenchymal phenotype. These results substantiate the idea that closely related but diverging neural crest-derived cell types respond selectively to TGFβ1 and demonstrate that these SK-N-SH-derived cell lines will be useful in experimental approaches that will allow us to infer mechanisms underlying regulation of neural crest differentiation.     

TGF-β receptor expression on human keratinocytes: A 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with type I and type II receptors

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-β has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-β receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-β receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-β1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-β receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-β1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-β receptors on keratinocytes, supporting the heterotetrameric model of TGF-β signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-β by markedly downregulating all four TGF-β binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with the TGF-β signalling receptors suggests that this GPI-anchored protein may modify TGF-β signalling in human keratinocytes. J. Cell. Biochem. 70:573–586, 1998. © 1998 Wiley-Liss, Inc.     

The Xenopus platelet-derived growth factor α receptor: cDNA Cloning and demonstration that mesoderm induction establishes the lineage-specific pattern of ligand and receptor gene expression

We have cloned the Xenopus PDGF α receptor cDNA and have used this clone, along with cDNA encoding PDGF A, to examine their expression pattern in Xenopus embryos and to determine the factors responsible for lineage specificity. Recombinant Xenopus α receptor expressed in COS cells exhibits PDGF-A-dependent tyrosine kinase activity. We find that receptor mRNA is present in cultured marginal zone tissue explants and in animal cap tissue induced to form mesoderm either by grafting to vegetal tissue or by treatment with recombinant activin A. In contrast, PDGF A mRNA is expressed in cultured, untreated animal cap tissue and is suppressed by mesoderm induction. These results suggest that ectodermally produced PDGF A may act on the mesoderm during gastrulation and that mesoderm induction establishes the tissue pattern of ligand and receptor expression. © 1993Wiley-Liss, Inc.     

A Recombinant Human TGF-β1 Fusion Protein with Collagen-Binding Domain Promotes Migration, Growth, and Differentiation of Bone Marrow Mesenchymal Cells

A continuous source of osteoblasts for normal bone maintenance, as well as remodeling and regeneration during fracture repair, is ensured by the mesenchymal osteoprogenitor stem cells of the bone marrow (BM). The differentiation and maturation of osteoprogenitor cells into osteoblasts are thought to be modulated by transforming growth factors-β (TGF-β1 and TGF-β2) and TGF-β-related bone morphogenetic proteins (BMPs). To define the responses of mesenchymal osteoprogenitor stem cells to several growth factors (GFs), we cultured Fischer 344 rat BM cells in a collagen gel medium containing 0.5% fetal bovine serum for prolonged periods of time. Under these conditions, survival of BM mesenchymal stem cells was dependent on the addition of GFs. Recombinant hTGF-β1-F2, a fusion protein engineered to contain an auxiliary collagen binding domain, demonstrated the ability to support survival colony formation and growth of the surviving cells, whereas commercial hTGF-β1 did not. Initially, cells were selected from a whole BM cell population and captured inside a collagen network, on the basis of their survival response to added exogenous GFs. After the 10-day selection period, the surviving cells in the rhTGF-β1-F2 test groups proliferated rapidly in response to serum factors (10% FBS), and maximal DNA synthesis levels were observed. Upon the addition of osteoinductive factors, osteogenic differentiation in vitro was evaluated by the induction of alkaline phosphatase (ALP) expression, the production of osteocalcin (OC), and the formation of mineralized matrix. Concomitant with a down-regulation of cell proliferation, osteoinduction is marked by increased ALP expression and the formation of colonies that are competent for mineralization. During the induction period, when cells organize into nodules and mineralize, the expression of OC was significantly elevated along with the onset of extracellular matrix mineralization. Differentiation of BM mesenchymal stem cells into putative bone cells as shown by increased ALP, OC synthesis, and in vitro mineralization required the presence of specific GFs, as well as dexamethasone (dex) and β-glycerophosphate (β-GP). Although rhTGF-β1-F2-selected cells exhibited the capacity to mineralize, maximal ALP activity and OC synthesis were observed in the presence of rhBMPs. We further report that a novel rhTGF-β1-F2 fusion protein, containing a von Willebrand's factor-derived collagen binding domain combined with a type I collage matrix, is able to capture, amplify, and stimulate the differentiation of a population of cells present in rat BM. When these cells are subsequently implanted in inactivated demineralized bone matrix (iDBM) and/or diffusion chambers into older rats they are able to produce bone and cartilage. The population of progenitor cells captured by rhTGF-β1-F2 is distinct from the committed progenitor cells captured by rhBMPs, which exhibit a considerably more differentiated phenotype.     

非小细胞肺癌患者血清CYFRA21-1、VEGF及CEA的表达及与临床病理特征的相关性研究

目的:研究非小细胞肺癌(NSCLC)患者血清细胞角蛋白19片段(CYFRA21-1)、血管内皮生长因子(VEGF)、癌胚抗原(CEA)的表达及与临床病理特征的相关性。方法:选取2015年12月至2016年4月在我院接受治疗的NSCLC患者120例作为观察组,另选取同期在我院接受治疗的肺部良性病变患者50例作为良性对照组,比较两组患者血清中CYFRA21-1、VEGF及CEA的表达,分析观察组患者血清中CYFRA21-1、VEGF及CEA的表达与临床病理特征之间的关系,采用Pearson相关系数分析观察组患者血清中CYFRA21-1、VEGF、CEA的相关性。结果:观察组患者血清中的CYFRA21-1、VEGF及CEA水平均高于良性对照组(P0.05)。鳞状细胞癌患者血清中CYFRA21-1水平高于腺癌患者,CEA水平低于腺癌患者(P0.05),鳞状细胞癌患者和腺癌患者血清中VEGF水平比较无统计学差异(P0.05)。TNM分期为III-IV期的观察组患者血清中CYFRA21-1、VEGF及CEA水平均明显高于I-II期患者,有统计学差异(P0.05)。经Pearson相关系数分析显示,观察组患者血清中CYFRA21-1与VEGF、CEA呈正相关(r=0.512,0.423,P=0.000,0.000),VEGF与CEA呈正相关(r=0.452,P=0.000)。结论:NSCLC患者血清中CYFRA21-1、VEGF及CEA呈高表达,且CYFRA21-1、CEA与病理类型和TNM分期有关,VEGF与TNM分期有关,且三种指标存在一定的相关性。     

The effect of coculture with human smooth muscle cells on the proliferation,the IL‐1 β secretion,the PDGF production and tube formation of human aortic endothelial cells

Endothelial cells (ECs) and smooth muscle cells (SMCs), which are the major component cells of blood vessels, produce various bioactive substances and communicate with each other through them. Although several studies of the interaction between ECs and SMCs have been reported, the effect of coculture with SMCs on ECs is still obscure. To clarify the interaction of ECs and SMCs, we examined the effect of coculture with SMCs on the proliferation, the IL‐1β secretion, the PDGF production and tube formation of ECs, using the coculture model: transferable wells and collagen gel. IL‐1 and PDGF are considered to be related to progression of atherosclerosis. Proliferation and tube formation of ECs are associated with repair of vessels. In the transferable well system coculture with SMCs stimulated the proliferation of ECs, and enhanced the IL‐1β secretion of ECs and in the collagen gel system coculture with SMCs induced the tube formation of ECs, and appeared to enhance the PDGF production of ECs. In conclusion, the effect of coculture with SMCs on ECs has two conflicting aspects: progression of atherosclerosis and angiogenesis. These results suggest that an imbalance of their effects may lead to pathological events. Copyright © 1999 John Wiley & Sons, Ltd.     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.