Role of matrix stiffness in the regulation of primary proximal tubular cell proliferation and differentiation:implication in chronic tubulointerstitial fibrosis

  • 陳 宛君

Student thesis: Doctoral Thesis


Normally differentiated renal proximal tubular epithelial cells (PTECs) reside on soft basement membrane containing laminin-rich extracellular matrix (ECM) Chronic tubulointerstitial fibrosis is characterized by the accumulation of collagen with tissue stiffening and finally leads to the end-stage renal disease Previous studies showed that transforming growth factor-beta 1 (TGF-beta1) played the potent initiator and/or enhancer for fibrogenesis However accumulated studies also indicate that the matrix stiffness also regulate cell behaviors i e development proliferation differentiation apoptosis and tumorigenesis We were curious about the role of matrix stiffness in the physiological and pathological development in the kidney In the first part of study we focused on the interplay between chemical cues and physical cues from ECM on the regulation of cell behaviors Primary culture of mice PTECs (mPTECs) gradually lost their tubular morphology and differentiated properties with the increase of cell spreading and proliferation when cultured on stiff matrix Furthermore the cells responded to TGF-beta1 and underwent epithelial-mesenchymal transition (EMT) under stiff matrix However these phenomena were reversed when cells were cultured on soft matrix On the other hand an increase of collagen amount in soft matrix also facilitated cell spreading de-differentiation proliferation and TGF-beta1-induced EMT indicating that both soft matrix and basement membrane signals were required for the maintenance of the physiological function of mPTECs Furthermore we identified that ERK activation controlled by stiff matrix contributed to all the cellular behaviors regulated by stiff matrix In the second part of study we attempted to verify the novel mechanism of how matrix stiffness controls cell proliferation and its significance on the pathogenesis of chronic tubulointerstitial fibrosis Based on the results obtained from oligo-microarray and experiments we purposed Kr?ppel-like factors 5 (Klf5) and Kr?ppel-like factors (Klf4) as the possible candidates Stiff matrix upregulated Klf5 and downregulated Klf4 in mRNA and protein levels via ERK signal Suppression of Klf5 or forced expression of Klf4 stunted stiff matrix-induced cell proliferation as confirmed by nuclear Cyclin D1 and EdU intensity indicating that Klf5/Klf4 served as the positive/negative regulators of cell proliferation respectively Furthermore we suggested that mechanosensitive Yes-associated protein 1 (YAP1) may contribute to stiff matrix induced Klf5 upregulation through preventing Klf5 degradation Finally we applied the in vivo model of unilateral ureteral obstruction to induce fibrosis Notably alleviation of tissue stiffening by blocking collagen crosslinker efficiently suppressed tubular dilatation and abnormal proliferation with the upregulation of ERK/YAP1/Klf5/Cyclin D1 axis and the downregulation of Klf4 In conclusion we demonstrate that how mechanical cues are involved in the regulation of renal physiological functions and pathological progression via mechanosensitive transcription factors This study provides us a new insight into the pathogenesis of chronic tubulointerstitial fibrosis from the physical view
Date of Award2015 Jan 26
Original languageEnglish
SupervisorMing-Jer Tang (Supervisor)

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