Cell metabolism is strongly influenced by mechano-environment. We show here that a fraction of kindlin-2 localizes to mitochondria and interacts with pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme for proline synthesis. Extracellular matrix (ECM) stiffening promotes kindlin-2 translocation into mitochondria and its interaction with PYCR1, resulting in elevation of PYCR1 level and consequent increase of proline synthesis and cell proliferation. Depletion of kindlin-2 reduces PYCR1 level, increases reactive oxygen species (ROS) production and apoptosis, and abolishes ECM stiffening-induced increase of proline synthesis and cell proliferation. In vivo, both kindlin-2 and PYCR1 levels are markedly increased in lung adenocarcinoma. Ablation of kindlin-2 in lung adenocarcinoma substantially reduces PYCR1 and proline levels, and diminishes fibrosis in vivo, resulting in marked inhibition of tumor growth and reduction of mortality rate. Our findings reveal a mechanoresponsive kindlin-2-PYCR1 complex that links mechano-environment to proline metabolism and signaling, and suggest a strategy to inhibit tumor growth.

Reprograming of proline metabolism is critical for tumor growth. Here we show that PINCH-1 is highly expressed in lung adenocarcinoma and promotes proline synthesis through regulation of mitochondrial dynamics. Knockout (KO) of PINCH-1 increases dynamin-related protein 1 (DRP1) expression and mitochondrial fragmentation, which suppresses kindlin-2 mitochondrial translocation and interaction with pyrroline-5-carboxylate reductase 1 (PYCR1), resulting in inhibition of proline synthesis and cell proliferation. Depletion of DRP1 reverses PINCH-1 deficiency-induced defects on mitochondrial dynamics, proline synthesis and cell proliferation. Furthermore, overexpression of PYCR1 in PINCH-1 KO cells restores proline synthesis and cell proliferation, and suppresses DRP1 expression and mitochondrial fragmentation. Finally, ablation of PINCH-1 from lung adenocarcinoma in mouse increases DRP1 expression and inhibits PYCR1 expression, proline synthesis, fibrosis and tumor growth. Our results identify a signaling axis consisting of PINCH-1, DRP1 and PYCR1 that regulates mitochondrial dynamics and proline synthesis, and suggest an attractive strategy for alleviation of tumor growth.

Pulmonary fibrosis is a progressive and fatal lung disease characterized by activation of lung fibroblasts and excessive deposition of collagen matrix. We show here that the concentrations of kindlin-2 and its binding partner PYCR1, a key enzyme for proline synthesis, are significantly increased in the lung tissues of human patients with pulmonary fibrosis. Treatment of human lung fibroblasts with TGF-β1 markedly increased the expression of kindlin-2 and PYCR1, resulting in increased kindlin-2 mitochondrial translocation, formation of the kindlin-2–PYCR1 complex, and proline synthesis. The concentrations of the kindlin-2–PYCR1 complex and proline synthesis were markedly reduced in response to pirfenidone or nintedanib, two clinically approved therapeutic drugs for pulmonary fibrosis. Furthermore, depletion of kindlin-2 alone was sufficient to suppress TGF-β1–induced increases of PYCR1 expression, proline synthesis, and fibroblast activation. Finally, using a bleomycin mouse model of pulmonary fibrosis, we show that ablation of kindlin-2 effectively reduced the concentrations of PYCR1, proline, and collagen matrix and alleviate the progression of pulmonary fibrosis in vivo. Our results suggest that kindlin-2 is a key promoter of lung fibroblast activation, collagen matrix synthesis, and pulmonary fibrosis, underscoring the therapeutic potential of targeting the kindlin-2 signaling pathway for control of this deadly lung disease.

Mesenchymal stem cells (MSCs) have been considered as ideal cells for the treatment of a variety of diseases. However, aging and spontaneous differentiation of MSCs during culture expansion dampen their effectiveness. Previous studies suggest that ex vivo aging of MSCs is largely caused by epigenetic changes particularly a decline of histone H3 acetylation levels in promoter regions of pluripotent genes due to inappropriate growth environment. In this study, we examined whether histone deacetylase inhibitor trichostatin A (TSA) could suppress the histone H3 deacetylation thus maintaining the primitive property of MSCs. We found that in regular adherent culture, human MSCs became flatter and larger upon successive passaging, while the expression of pluripotent genes such as Oct4, Sox2, Nanog, Rex-1, CD133 and TERT decreased markedly. Administration of low concentrations of TSA in culture significantly suppressed the morphological changes in MSCs otherwise occurred during culture expansion, increased their proliferation while retaining their cell contact growth inhibition property and multipotent differentiation ability. Moreover, TSA stabilized the expression of the above pluripotent genes and histone H3 acetylation levels in K9 and K14 in promoter regions of Oct4, Sox2 and TERT. Our results suggest that TSA may serve as an effective culture additive to maintain the primitive feature of MSCs during culture expansion.

Introduction: Rat middle cerebral artery occlusion (MCAO) model is the most commonly used animal model in ischemic stroke studies. In the model, to increase the amount of stem cells or drugs to enter the brain after delivery into the internal carotid artery (ICA), the pterygopalatine artery (PPA) is occluded. However, PPA occlusion is a technically demanding procedure which often causes complications. Methods: In this study, we developed an ICA injection needle to facilitate easy and efficient delivery of stem cells to the ischemic brain through the ICA without the need of PPA occlusion. We injected methylene blue and fluorescence dye DiI-labeled human mesenchymal stem cells (DiI-hMSCs) into the ICA in rats with the ICA injection needle (without PPA ligation) or the conventional micro-injection needle (with PPA ligation) and assessed their distributions. Results: When methylene blue was injected, evident blue stains were found in the brain of the injection side particularly the middle cerebral artery (MCA)-supplied areas but not in the PPA supplied areas. Similarly, when DiI-hMSCs were injected, the cells largely appeared in the MCA-supplied tissues, which were similar in quantity compared to conventional micro-injection needle injection with PPA occlusion. Moreover, hMSCs injected with the ICA needle or the micro-injection needle similarly improved the functional recovery of the infarcted brain. Conclusions: Our results indicate that the ICA injection needle is easy to use and efficient in delivering cells to the ischemic brain tissue in rat MCAO model.

Mechano-environment plays multiple critical roles in the control of mesenchymal stem cell (MSC) fate decision, but the underlying signaling mechanisms remain undefined. We report here a signaling axis consisting of PINCH-1, SMAD specific E3 ubiquitin protein ligase 1 (Smurf1), and bone morphogenetic protein type 2 receptor (BMPR2) that links mechano-environment to MSC fate decision. PINCH-1 interacts with Smurf1, which inhibits the latter from interacting with BMPR2 and consequently suppresses BMPR2 degradation, resulting in augmented BMP signaling and MSC osteogenic differentiation (OD). Extracellular matrix (ECM) stiffening increases PINCH-1 level and consequently activates this signaling axis. Depletion of PINCH-1 blocks stiff ECM-induced BMP signaling and OD, whereas overexpression of PINCH-1 overrides signals from soft ECM and promotes OD. Finally, perturbation of either Smurf1 or BMPR2 expression is sufficient to block the effects of PINCH-1 on BMP signaling and MSC fate decision. Our findings delineate a key signaling mechanism through which mechano-environment controls BMPR2 level and MSC fate decision.