Extracellular collagen modulates the regulation of chondrocytes by transforming growth factor-β1

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-β1, Type-I and type-II collagen, heat denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-β1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [³⁵S]sulfate and [³H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-β1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-β1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-β1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-β1.     

Effects of transforming growth factor-β1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I

The local tissue metabolism is controlled through the complex interaction between systemic and local growth factors. In recent years, an increasing number of autocrine or paracrine growth regulators have been identified in physeal cartilage. While these factors act to alter chondrocytes phenotypically and presumably are important mediators in the process of endochondral ossification, the manner in which they interact with the systemically regulated growth factor insulin-like growth factor-I is unknown. In the present study, the interactive effects of insulin-like growth factor-I with transforming growth factor-β1 or basic fibroblast growth factor were examined in short-term monolayer cultures of chick growth plate chondrocytes. [³H]thymidine incorporation was maximally stimulated 11-fold by fibroblast growth factor (10 ng/ml) and 3.5-fold by transforming growth factor-β1 following a 24-hour exposure in serum-containing cultures. The effects of transforming growth factor-β1 and fibroblast growth factor at both high and low concentrations were enhanced in a dose-dependent manner by insulin-like growth factor-I, with a 40–50% increase in DNA synthesis in the presence of 100 ng/ml of insulin-like growth factor-I. Since insulin-like growth factor-I increased [³H]thymidine incorporation after 48 hours (50% increase) but not after 24 hours of exposure, these observations represent a synergistic interaction. Total DNA in cultures treated for 5 days confirmed the modulating effect of insulin-like growth factor-I with transforming growth factor-β1 and fibroblast growth factor. The growth factors were further examined for their effects on markers of chondrocyte differentiation. While all three caused a dose-dependent inhibition of alkaline phosphatase activity, the effects of insulin-like growth factor-I were additive only to those of transforming growth factor-β1 and fibroblast growth factor. Similarly, insulin-like growth factor-I did not affect the sulfate incorporation stimulated by fibroblast growth factor or transforming growth factor-β1. Insulin-like growth factor-I had no effect on total protein synthesis after 24 hours and, although type-II collagen mRNA levels were stimulated, it had no effect on type-X collagen mRNA, as determined by quantitative in situ hybridization. Finally, insulin-like growth factor-I did not alter the dose-dependent stimulation of noncollagen protein synthesis and the inhibition of collagen synthesis caused by fibroblast growth factor and transforming growth factor-β1 in 24-hour cultures. Thus, the data suggest that insulin-like growth factor-I may have a role in augmenting the effects of other growth factors found in cartilage. Since insulin-like growth factor-I is under systemic control by growth hormones, this permits an endocrine regulation of transforming growth factor-β1 and fibroblast growth factor activity and may bring local growth factor effects under systemic control.     

Differentiated cellular function in fetal chondrocytes cultured with insulin-like growth factor-I and transforming growth factor-β

This study examined fetal chondrocyte proliferation and function following exposure to transforming growth factor-β and insulin-like growth factor-I. Fetal equine articular chondrocytes of the early third-trimester were isolated and cultured in monolayer conditions, then exposed to 0,1,5, or 10 ng/ml transforming growth factor-β or 0,10,50, or 100 ng/ml insulin-like growth factor-I for 48 hours. Proliferative responses were assessed by cell counts and [³H]thymidine uptake into precipitable DNA. Differentiated chondrocyte metabolic activity was determined by sulfated glycosaminoglycan quantitation, ³⁵[SO₄] incorporation into precipitable glycosaminoglycan, and proteoglycan molecular sizing by CL-2B column chromatography. Morphological changes seen on phase-contrast microscopy included a larger proportion of rounded cells in monolayer cultures supplemented with insulin-like growth factor-I and cytotoxic changes in cells treated with transforming growth factor-β. Both insulin-like growth factor-I and transforming growth factor-β resulted in significant elevations of [³H]thymidine uptake; however, cell numbers did not rise sufficiently over the 48-hour culture period to reach significant levels. Maximum mitogenic responses were evident at 50 and 100 ng/ml insulin-like growth factor-I and 5 ng/ml transforming growth factor-β. The production of proteoglycan was also enhanced (435%) by exposure to 50 ng/ml insulin-like growth factor-I, and an increased proportion of larger proteoglycan monomer species was evident in cultures treated with 50 and 100 ng/ml insulin-like growth factor-I. A similar dose-response was also evident in cultures treated with transforming growth factor-β (maximal 164%' increase with 5 ng/ml), although the presence of serum in the culture medium altered the pattern of enhanced proteoglycan synthesis to favor the lower concentration of 1 ng/ml (191%). Additionally larger proteoglycan molecules were synthesized in response to high concentrations of transforming growth factor-β in serum-free cultures. Significant biochemical changes resulted from the addition of transforming growth factor-β to fetal chondrocyte cultures; however, monolayer cultures that were treated with transforming growth factor-β and supplemented with serum began to develop cellular toxicity, including nuclear pyknosis and cytoplasmic fragmentation. Degenerative cellular changes were not evident in cultures treated with insulin-like growth factor-I, and significant differentiated metabolic activity resulted from the presence of insulin-like growth factor-I in the culture medium. These data suggest that the responses of fetal chondrocytes to insulin-like growth factor-I and transforming growth factor-β were enhanced compared with the responses of chondrocytes derived from postnatal animals and that these metabolically active cells can be primed by endogenous or exogenous growth factors to provide enhanced articular function and repair.     

Proliferative and matrix synthesis response of canine anterior cruciate ligament fibroblasts submitted to combined growth factors

We investigated the effects of growth factors on the proliferation and matrix synthesis of anterior cruciate ligament fibroblasts. Fibroblasts from the anterior cruciate ligaments of dogs were transferred at the second passage in a defined medium. Epidermal growth factor, platelet-derived growth factor-AB, transforming growth factor-β₁, insulin-like growth factor-1, and insulin, combined two by two following a 5 × 5 logarithmic concentration matrix, were added. Tridimensional curves showing cell proliferation at 24 hours against the concentration of two effectors were obtained for each combination. Collagen and proteoglycan productions were quantified using [¹⁴ C]glycine and Na₂[³⁵S]O₄. Ratios of type I:III collagen and hydrodynamic size distributions of proteoglycans were assayed, respectively, by sodium dodecyl sulfate-polyacrylamidel gel electrophoresis and gel filtration chromatography. Epidermal growth factor had an effect nearly equivalent to that of platelet-derived growth factor-AB on cell proliferation. Both had a greater effect than insulin-like growth factor-1, which in turn had a greater effect than both the effect of insulin or the nearly equivalent effect of transforming growth factor-β₁. Neither platelet-derived growth factor-AB nor insulin has a significant effect by itself on collagen production. Epidermal growth factor slightly decreases collagen production as well as the type I:III collagen ratio: both transforming growth factor-β₁ and insulin-like growth factor-1 increase the same parameters. Epidermal growth factor inhibits the stimulation induced by transforming growth factor-β₁. Similarly, insulin decreases the response to insulin-like growth factor-1. Proteoglycan production was significantly increased by all growth factors in this study, with transforming growth factor-β₁ having the strongest effect, Small hydrodynamic size of proteoglycan was correlated to a high level of proteoglycan biosynthesis. The results may be readily applied to tissue engineering or provide a basis for in vivo investigations.     

Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion,and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs

The present study was undertaken to investigate the effect of insulin-like growth factor-1 on proteoglycan synthesis and the autocrine/paracrine mechanisms involving insulin-like growth factor-1 in the bovine coccygeal intervertebral disc. Insulin-like growth factor-1 stimulated proteoglycan synthesis in cultured cells of the nucleus pulposus of bovine intervertebral discs in a dose-dependent manner, and the effect was inhibited by an anti-insulin-like growth factor-1 monoclonal antibody. In situ hybridization histochemistry revealed the expression of insulin-like growth factor-1 mRNA in the cultured cells, and its production in these cells was demonstrated by radioimmunoassay. Insulin-like growth factor-1 receptor in the cultured cells was also demonstrated immunohistochemically. Scatchard analysis using an [¹²⁵I]insulin-like growth factor-1 binding assay showed that the cells cultured in monolayer had a single type of insulin-like growth factor-1 receptor, whose affinity and number were estimated to be 7.38 × 10⁸/M and 9.27 × 10⁴/cell, respectively. These results suggest that insulin-like growth factor-1 stimulates proteoglycan synthesis in cells of the nucleus pulposus and that these cells in culture have an insulin-like growth factor-1 autocrine/paracrine mechanism. The expressions of insulin-like growth factor-1 mRNA and insulin-like growth factor-1 receptor in disc tissue were greater in cells of the nucleus pulposus of fetal bovine intervertebral discs than in those of the adult discs. These findings suggest that the action of autocrine/paracrine insulin-like growth factor-1 is more active in cells of the young nucleus pulposus than in cells of mature subjects.     

TGF-β1和TGF-α在膀胱癌侵袭和转移中的作用

目的 探讨转化生长因子-β（TGF-β1）和转化生长因子－α（TGF-α）对膀胱癌细胞株（EJ）生长的影响以及促进膀胱癌侵袭和转移的可能途径。方法 采用MTT、Western Blot和RT－PCR方法,观察TGF－β1和TGF－α对DJ细胞株生长以及在mRNA和蛋白水平上对金属基质蛋白酶（MMPs)和组织特异性金属蛋白酶抑制剂－2（TIMP－2）表达的影响。结果 （1）TGF-β1和TGF－α对EJ细胞生长存在抑制趋势,但差别无显著性意义。（2）TGF－β1和TGF－α作用于EJ细胞48h时 ,MMP－2、TIMP－2、MT1－MMP mRNA表达降低;TGF－β1组MMP-9 mRNA表达增加,而对照组和TGF－α组无MP－9mRNA表达。（3）当TGF－β1浓度为0．1、1．0ng/ml时,增加细胞培养上清液中MMP－2蛋白含量对TIMP－2蛋白水平无影响。当浓度为5．0、10．0ng/ml时对MMP－2无影响而降低TIMP－2表达;TGF－α在浓度为1.0、5.0、10.0ng/ml时,对MMP－2蛋白表达无影响而降低TIMP－2表达;在100．0ng/ml时增加MMP－2蛋白表达而对TIMP－2表达无影响;无论在对照组和实验组均未检测到MMP－9。结论 TGF-β1、TGF-α不能抑制EJ细胞生长,参与肿瘤侵袭和转移作用可能与调节MMPs、TIMP－2表达途径有关。     

Effects of basic fibroblast growth factor,transforming growth factor-β1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants

This study evaluated the effects of basic fibroblast growth factor, transforming growth factor-β1, insulin-like growth factor-1, and insulin on the incorporation of thymidine and sulfate in human osteoarthritic articular cartilage. Tissue explants were obtained from 11 patients undergoing total knee arthroplasty and were categorized as nonfibrillated or fibrillated cartilage. The explants were cultured for 22 days, with changes of medium and growth factor every 72 hours, and labeled with [³H]thymidine and [³⁵S]sulfate. Growth factors were used in the following concentrations: basic fibroblast growth factor at 1, 10, and 100 ng/ml; transforming growth factor-β1 at 0.5, 5, and 50 ng/ml; insulin-like growth factor-1 at 0.15, 1.5, and 15 ng/ml; and insulin at 0.05, 0.5, and 5 μg/ml. Basic fibroblast growth factor decreased thymidine incorporation to 70% and sulfate incorporation to less than 20% that of the growth factor-free controls. Transforming growth factor-β1 had no significant effect on thymidine incorporation, whereas the concentrations studied inhibited sulfate incorporation to approximately 40% that of the controls. At the concentrations tested, insulin-like growth factor-1 had no significant effect on incorporation of either thymidine or sulfate. In contrast, insulin significantly stimulated the incorporation of both. Compared with growth factor-free controls, insulin maximally increased thymidine incorporation by a factor (± EM) of 2.36 ± 0.47 and 1.69 ± 0.19 in nonfibrillated and fibrillated explants, respectively; sulfate incorporation was maximally increased 1.60 ± 0.24 and 1.92 ± 0.29-fold for nonfibrillated and fibrillated explants, respectively. Of the factors tested, insulin demonstrated the greatest promise for promoting a synthetic response that may contribute to the regeneration of osteoarthritic cartilage.     

Responsiveness of bovine chondrocytes to growth factors in medium with different serum concentrations.

Autologous transplantation of chondrocytes is currently under investigation as a potential therapy to stimulate intrinsic repair in articular cartilage defects. The quality of the repair tissue may benefit from the preservation of the characteristic chondrocytic phenotype of the transplanted cells together with the production of a new extracellular matrix composed of collagen type II and larger proteoglycans. A number of growth factors are believed to play an important role in the process of generating new cartilage repair tissue. In this study, the dose-dependent response of bovine chondrocytes to recombinant human insulin-like growth factor-1, recombinant human transforming growth factor-beta2, and recombinant human bone morphogenetic protein-2 was studied in an alginate culture system under different culture conditions. The chondrocytes were cultured in medium with increasing concentrations of fetal calf serum. The cultures were assessed by the total amount of DNA, quantitative and qualitative synthesis of proteoglycan, production of nitric oxide, and histology. Cells cultured in the presence of each growth factor had an equal, nonsignificant stimulation of DNA synthesis compared with those cultured in basal medium alone. Recombinant human insulin-like growth factor-1 and recombinant human transforming growth factor-beta2 stimulated proteoglycan synthesis in a dose-dependent and reversed dose-dependent fashion, respectively. Recombinant human bone morphogenetic protein-2 stimulated proteoglycan synthesis significantly only in the absence of fetal calf serum or in the presence of small amounts of the serum. Overall, proteoglycan synthesis dramatically decreased with the addition of each growth factor as the concentration of fetal calf serum in the medium decreased, and the dose-dependent stimulation pattern, as observed for recombinant human insulin-like growth factor-1 and recombinant human transforming growth factor-beta2, disappeared. Apart from a moderate increase in mRNA for aggrecan and decorin, the growth factors did not greatly affect the type of proteoglycans synthesized. Histological examination confirmed the presence of a dense pericellular matrix deposition, especially when the chondrocytes were cultured in the presence of recombinant human insulin-like growth factor-1 or recombinant human transforming growth factor-beta2. The results indicate that these growth factors can stimulate qualitatively superior matrix production and that the responsiveness of the chondrocytes to the growth factors changes with the culture conditions. Further knowledge about the interaction between chondrocytes, growth factors, and the external environment is important to stimulate chondrocytes to produce adequate repair tissue in cartilage defects in vivo. Insulin-like growth factor-1 especially seems capable of stimulating, in the most consistent and predictable fashion, qualitatively superior proteoglycan synthesis by differentiated chondrocytes. Additional in vivo studies are needed to evaluate the potential of these growth factors as stimulators in cartilage repair.     

Transforming growth factor-β1 and fibroblast growth factors in rat growth plate

Chondrocytes in the growth plate progress in an orderly fashion from resting through proliferating to hypertrophic cells. In the region of hypertrophic chondrocytes, the cartilage is invaded by capillary loops and endochondral ossification is initiated. It is currently believed that growth factors may regulate the proliferation and maturation of chondrocytes and the synthesis of extracellular matrix in the growth plate. The ordered sequence of proliferation and differentiation observed in the growth plate provides a unique opportunity to study the role of acidic fibroblast growth factor, basic fibroblast growth factor, and transforming growth factor-β1 in the regulation of these processes. In this study, expression of the mRNA of these growth factors was examined using total RNA extracted from the physis and epiphysis of rat tibias. Transforming growth factor-β1 mRNA was detected by Northern hybridization. Expression of the genes encoding acidic and basic fibroblast growth factors was demonstrated by polymerase chain reaction amplification. In addition, using polyclonal antibodies against these growth factors, we localized them by immunohistochemical analysis. Strong intracellular staining with a predominantly nuclear pattern was observed in chondrocytes from the proliferating and upper hypertrophic zones. In contrast, chondrocytes in the resting zone stained only faintly for the presence of these growth factors. Some chondrocytes in the resting zone adjacent to the proliferating zone stained with these antibodies, and the antibodies also stained cells in the zone of Ranvier, which regulates latitudinal bone growth. Lastly, the location of transforming growth factor-β1 was examined further with use of a polyclonal antipeptide antibody specific for its extracellular epitope. Interestingly, extracellular staining for transforming growth factor-β1 was observed only around chondrocytes in the hypertrophic zone. These results suggest a role for these growth factors in the regulation of proliferation and maturation of chondrocytes and in endochondral ossification.     

Expression of cartilage extracellular matrix and potential regulatory genes in a new human chondrosarcoma cell line

A human chondrosarcoma cell line has been established from an aggressive chondrosarcoma. The cells grow in a monolayer culture (doubling time: 2 days) and form aggregates. The aggregates consist of a rim of cells surrounding a hollow core. The cell line exhibits a unique pattern of mRNA expression with several molecules characteristic of the chondrocyte phenotype. Consistent with the chondrocyte phenotype, mRNAsencoding types IX and XI collagens were present along with an abundant expression of mRNAsencoding the core protein of the cartilage proteoglycans biglycan and aggrecan. No expression of mRNAs encoding types I or II fibrillar collagens or the proteoglycan decorin was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of [³⁵S]sulfate-radiolabeled material confirmed the translation of proteoglycans containing glycosaminoglycan chains. The expression of molecules that contribute to cartilage development and tumorigenesis was examined. The cell line produces abundant mRNA that encodes transforming growth factor-β1, a member of a family of cartilage and bone inductive proteins. The expression of mRNA encoding two proteins associated specifically with chondrogenesis was detected: Cart-1, a homeobox protein involved in cartilage differentiation, and CD-RAP, a secreted molecule restricted under normal conditions to differentiating chondrocytes and cartilage. Overexpression of p53, a tumor-suppressor gene, was detected. DNA analysis revealed a loss of heterozygosity at the chromosomal locus encoding p53, with the deletion of one p53 allele and the mutation of the remaining allele in both the parent tumor and the cell line. The malignant chondrosarcoma phenotype may be related to the unique gene expression pattern that is characteristic in many ways of differentiating chondroblasts, as well as to the inactivation of the p53 function that could contribute to the proliferative capacity of the cell line. This cell line may serve as a biological model for further investigation of the etiology of human chondrosarcomas and for the synthesis and regulation of cartilage-specific genes.     

Osteosarcoma oncogene expression detected by in situ hybridization

Fifteen archival human osteosarcoma specimens were examined by in situ hybridization for the expression of human and mouse transforming growth factor-β (isoforms 1, 2, and 3), c-fos, and metalloproteinase (stromelysin-3 and matrilysin). Osteosarcoma subtypes were confirmed by review of patients' radiographs, histopathology, and age at diagnosis. The outcome and method of treatment were documented. The subtypes of osteosarcoma consisted of nine conventional osteosarcomas and two each of fibroblastic, telangiectatic, and post-radiation osteosarcomas. Each specimen was histologically examined under light microscopy, and then adjacent paraffin sections were assayed with sense and anti-sense RNA probes by in situ hybridization. The probes localized to the neoplastic cells, confirming the methodology of the technique. Human transforming growth factor-β1 had the most uniform binding affinity to the osteosarcomas examined and was more specific in binding than mouse transforming growth factor-β1. Specific mRNA encoding for the transforming growth factor-βs, c-fos, and metalloproteinases are detectable in patterns within osteosarcoma cells, and collectively, their expression parallels the different histopathologic subtypes. The less differentiated subtypes (telangiectatic and post-radiation osteosarcomas) expressed the fewest molecular markers. Osteosarcoma is a heterogeneous tumor. Differential expression of matrilysin in osteosarcoma is the first reported detection of metalloproteinase activity in human skeletal sarcoma.     

肾康注射液拮抗马兜铃酸对人近端肾小管上皮细胞作用的研究

目的：探讨肾康注射液（SKI）能否拮抗马兜铃酸钠盐（AA-Na）诱发的人近端肾小管上皮细胞（HKC）的促纤维化效应。方法：AA-Na（10mg/L）加或不加SKI（8mg/ml）与HKC孵育,然后检测转化生长因子-β1（TGF-β1）、结缔组织生长因子（CTGF）、金属蛋白酶组织抑制物-1（TIMP-1）及纤溶酶原激活物抑制物-1（PAI-1）的mRNA表达（孵育12h,RT-PCR方法检测）和蛋白质表达（孵育36h用免疫印迹法检测胞内CTGF,孵育24h用ELISA法检测上清中其他因子）。结果：AA-Na能显著上调HKC对TGF-β1、CTGF、TIMP-1、PAI-1的表达,与对照组比较,mRNA表达分别上调1.84,1.58,1.62,1.29倍,蛋白质表达分别上调1.12,1.63,1.42,1.29倍,P均〈0.05;加SKI后,上述因子的高表达均被显著抑制,与AA-Na组比较,mRNA表达的抑制率分别为41.6%,43.7%,43.8%及24.4%,蛋白质表达的抑制率分别为34.3%,43.3%,31.1%及21.9%,P均〈0.05。结论：AA-Na能刺激HKC显著上调促细胞外基质（ECM）合成因子（TGF-β1、CT-GF）及抗ECM降解因子（TIMP-1、PAI-1）的mRNA及蛋白质表达,而SKI能拮抗AA-Na的上述作用。     

LeftyA真核表达载体的构建及其对转化生长因子-β1诱导人肾小管上皮细胞转分化的影响

目的 构建pcDNA3.1-LeftyA真核表达载体,观察其对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞(HK-2)转分化(EMT)的影响.方法 基因克隆技术构建pcDNA3.1-LeftyA真核表达载体,将其瞬时转染HK-2细胞,TGF-β1(10μg/L)刺激后,观察细胞形态变化,检测E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)基因及蛋白的表达.结果 TGF-β1刺激后HK-2细胞内E-cadherin mRNA和蛋白时间依赖性表达下调,α-SMA mRNA和蛋白时间依赖性表达上调,同时E-cadherin表达变化早于α-SMA;LeftyA蛋白可以显著抑制E-cadherin蛋白的下调表达(P＜0.05),其表达比同时间点单纯刺激组高17.6%;同时可逆转α-SMA蛋白的上调表达(P＜0.05),其蛋白表达比单纯刺激组低14.0%.结论 LeftyA蛋白可以抑制TGF-β1所致的EMT.

Abstract：

Objective To construct the eukaryotic expression vector for LeftyA and study the effects of LeftyA on epithelial to mesenchymal transition of human proximal tubular epithelial cells induced by transforming growth factor-β1 ( TGF-β1 ). Methods The pcDNA3. 1-LeftyA was constructed by recombinant DNA technique. After transfection with pcDNA3. 1-LeftyA HK-2 cells were stimulated by TGF-β1( 10 μg/L). The morphological changes, and the expression of E-cadherin, α-SMA and LeftyA mRNA and protein were observed and detected, respectively. Results TGF-β1 could markedly decrease the expression of E-cadherin mRNA and protein in HK-2 cells induced, and dramatically increase the expression of α-SMA mRNA and protein in a time-dependent manner. Forced expression of exogenous LeftyA led to a blockage of TGF-β1 -induced E-cadherin ( 17.6% ) suppression and α-SMA induction ( 14. 0% ). Conclusion Disruption of cell adherence is the beginning stage of EMT, and overexpression of LeftyA can suppress EMT induced by TGF-β1, which suggests a alprostadil role for LeftyA in TGF-β1-induced tubular EMT and renal fibrosis.     

虫草菌液拮抗马兜铃酸对人近端肾小管上皮细胞的作用

目的 探讨虫草菌液能否拮抗马兜铃酸诱发的人近端肾小管上皮细胞系(HKC)的促纤维化效应。 方法 马兜铃酸钠盐(AA-Na，40 mg/L)加或不加虫草菌液(10 mg/L)与HKC孵育(孵育12 h检测mRNA表达，孵育36 h检测蛋白质表达)，然后检测HKC中转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、金属蛋白酶1组织抑制物(TIMP-1)以及纤溶酶原激活物抑制物1(PAI-1)的mRNA(RT-PCR法)和相应蛋白质(CTGF表达采用免疫印迹法，其余采用ELISA方法)表达。结果 AA-Na能显著上调HKC对TGF-β1、CTGF、TIMP-1、PAI-1的表达，与对照组比较，mRNA表达分别上调1.24、1.31、1.27及1.36倍，蛋白质表达分别上调2.50、1.75、2.13及1.46倍，P均<0.05。加虫草菌液后，上述TGF-β1、CTGF及TIMP-1的高表达被显著抑制，与AA-Na组比较，mRNA表达的抑制率分别为12%、20%及17%；蛋白质表达的抑制率分别为25%、20%及37%，P均<0.05，但未能抑制PAI-1的高表达(P > 0.05)。结论 虫草菌液可下调AA-Na刺激的HKC促细胞外基质(ECM)合成因子(TGF-β1、CTGF)及抗ECM降解因子(TIMP-1)的表达。     

The effect of hydrostatic pressure on intervertebral disc metabolism.

STUDY DESIGN: By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in an alginate. OBJECTIVE: To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells. SUMMARY OF BACKGROUND DATA: The influence of compression (both hydrostatic and mechanical) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc, was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate. METHODS: Fresh cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 1 MPa and the other at atmospheric pressure. The effects of 1 MPa were compared against atmospheric pressure by measuring the incorporation of [3H]-proline and [35S]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and II collagen. RESULTS: Comparisons with atmospheric pressure yielded the following findings: 1) In the incorporation studies, the nucleus and anulus cells exhibited a differential response to a hydrostatic pressure of 1 MPa. Collagen and proteoglycan syntheses were stimulated in the nucleus cells and inhibited in the anulus cells. 2) There was no significant increase in cell proliferation, as measured by DNA content, at 1 MPa for either the anulus or nucleus cells. 3) The mRNA levels of collagen (Col 1A1 and Col 2A1) and aggrecan increased at 1 MPa in both the nucleus and anulus cells. CONCLUSIONS: Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells.     

Growth differentiation factor-15: induction in liver injury through p53 and tumor necrosis factor-independent mechanisms

Expression of macrophage inhibitory cytokine-1 (MIC-1), a divergent transforming growth factor-beta family member, and its murine ortholog, growth/differentiation factor-15 (GDF-15), is induced in hepatocytes by surgical and chemical injury and heat shock. Here, we demonstrate that the regulation of GDF-15/MIC-1 expression may be evolutionarily conserved because MIC-1 was induced in diseased human livers. Gdf15 induction was independent of protein synthesis, a hallmark of immediate-early gene regulation. Although tumor necrosis factor (TNF) induced GDF-15 expression, injury-elicited Gdf15 expression was not reduced in mice deficient for both TNF receptor subtypes. Furthermore, although the stress sensor p53 is known to induce GDF-15/MIC-1 expression, injury-elicited Gdf15 expression was unchanged in p53 null mice. Our results demonstrate that GDF-15 induction is an immediate early response to liver injury that can occur through TNF and p53 independent pathways.