Altieri P, Zegarra Moran O, Galietta L J, Tarelli L T, Sessa A, Ghiggeri G M
Section of Nephrology, G. Gaslini Children Hospital, Genoa, Italy.
J Cell Physiol. 1998 Nov;177(2):214-23. doi: 10.1002/(SICI)1097-4652(199811)177:2<214::AID-JCP3>3.0.CO;2-Q.
Transforming growth factor beta (TGFbeta) determines a nearly complete inhibition of cystogenesis by MDCK cells grown in collagen I-enriched matrices in vitro. In order to elucidate the mechanism implicated in this phenomenon, we performed a series of experiments aimed at discovering a relevant role of extracellular matrix. TGFbeta (2 ng/ml) played a marked stimulatory effect on the expression of extracellular matrix by MDCK with a selective effect on collagen V (three to fourfold increase of protein and mRNA) and in parallel inhibited cystogenesis by 95%. Cotreatment with TGFbeta and anti-collagen V antibodies restored a normal cystogenesis. In analogy, when MDCK cells were grown in three-dimensional matrices containing collagen I and minor (10%) amounts of collagen V, cystogenesis was once again inhibited by 95%. To characterize the molecular mechanism activated by TGFbeta and collagen V, we looked at the electrophysiological characteristics of MDCK monolayers and found a drastic fall of transepithelial electrical resistance (TER) in both conditions. In parallel with the decrease in TER, TGFbeta and collagen V also induced the leakage of two high molecular weight tracers, i.e., [3H]-inulin and 150 kD FITC-Dextran, suggesting a perturbation of the paracellular permeability. Finally, TGFbeta at the relevant concentration did not stimulate apoptosis in our cellular model, as judged by propidium iodide staining and by in situ end labeling of DNA fragments. These observations suggest that TGFbeta inhibits cystogenesis by MDCK cells in vitro by altering the collagenic composition of the three-dimensional milieu where MDCK cells grow and form cysts. The molecular mechanism responsible for inhibition of cystogenesis is the increase of paracellular flux which overcomes the active transport of solutes and water inside cysts.
转化生长因子β（TGFβ）可使体外在富含I型胶原的基质中生长的MDCK细胞的囊肿形成几乎完全受到抑制。为了阐明这一现象背后的机制，我们进行了一系列实验，旨在发现细胞外基质的相关作用。TGFβ（2 ng/ml）对MDCK细胞外基质的表达具有显著的刺激作用，对V型胶原具有选择性作用（蛋白质和mRNA增加三到四倍），同时将囊肿形成抑制了95%。TGFβ与抗V型胶原抗体共同处理可恢复正常的囊肿形成。类似地，当MDCK细胞在含有I型胶原和少量（10%）V型胶原的三维基质中生长时，囊肿形成再次被抑制了95%。为了表征TGFβ和V型胶原激活的分子机制，我们研究了MDCK单层的电生理特性，发现在这两种情况下跨上皮电阻（TER）都急剧下降。与TER降低同时，TGFβ和V型胶原还诱导了两种高分子量示踪剂即[3H] - 菊粉和150 kD FITC - 葡聚糖的渗漏，提示细胞旁通透性受到干扰。最后，通过碘化丙啶染色和DNA片段原位末端标记判断，相关浓度的TGFβ在我们的细胞模型中并未刺激细胞凋亡。这些观察结果表明，TGFβ通过改变MDCK细胞生长和形成囊肿的三维环境中的胶原组成，在体外抑制MDCK细胞的囊肿形成。抑制囊肿形成的分子机制是细胞旁通量增加，这超过了溶质和水在囊肿内的主动转运。
