α-parvin controls chondrocyte column formation and regulates long bone development.

Endochondral ossification requires proper control of chondrocyte proliferation, differentiation, survival, and organization. Here we show that knockout of α-parvin, an integrin-associated focal adhesion protein, from murine limbs causes defects in endochondral ossification and dwarfism. The mutant long bones were shorter but wider, and the growth plates became disorganized, especially in the proliferative zone. With two-photon time-lapse imaging of bone explant culture, we provide direct evidence showing that α-parvin regulates chondrocyte rotation, a process essential for chondrocytes to form columnar structure. Furthermore, loss of α-parvin increased binucleation, elevated cell death, and caused dilation of the resting zones of mature growth plates. Single-cell RNA-seq analyses revealed alterations of transcriptome in all three zones (i.e., resting, proliferative, and hypertrophic zones) of the growth plates. Our results demonstrate a crucial role of α-parvin in long bone development and shed light on the cellular mechanism through which α-parvin regulates the longitudinal growth of long bones.

Kindlin-2 Acts as a Key Mediator of Lung Fibroblast Activation and Pulmonary Fibrosis Progression.

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-ß1 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-ß1-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.

PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and 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.

A PINCH-1-Smurf1 signaling axis mediates mechano-regulation of BMPR2 and stem cell differentiation.

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.

TSA restores hair follicle-inductive capacity of skin-derived precursors.

The genesis of the hair follicle relies on signals derived from mesenchymal cells in the dermis during skin morphogenesis and regeneration. Multipotent skin-derived precursors (SKPs), which exhibit long term proliferation potential when being cultured in spheroids, have been shown to induce hair genesis and hair follicle regeneration in mice, implying a therapeutic potential of SKPs in hair follicle regeneration and bioengineering. However, the hair-inductive property of SKPs declines progressively upon ex vivo culture expansion, suggesting that the expressions of the genes responsible for hair induction are epigenetically unstable. In this study, we found that TSA markedly alleviated culture expansion induced SKP senescence, increased the expression and activity of alkaline phosphatase (AP) in the cells and importantly restored the hair inductive capacity of SKPs. TSA increased the acetylation level of histone H3, including the K19/14 sites in the promoter regions of bone morphogenetic proteins (BMPs) genes, which were associated with elevated gene expression and BMP signaling activity, suggesting a potential attribution of BMP pathway in TSA induced recovery of the hair inductive capacity of SKPs.

Kindlin-2 links mechano-environment to proline synthesis and tumor growth.

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.

Kindlin-2 regulates mesenchymal stem cell differentiation through control of YAP1/TAZ.

Precise control of mesenchymal stem cell (MSC) differentiation is critical for tissue development and regeneration. We show here that kindlin-2 is a key determinant of MSC fate decision. Depletion of kindlin-2 in MSCs is sufficient to induce adipogenesis and inhibit osteogenesis in vitro and in vivo. Mechanistically, kindlin-2 regulates MSC differentiation through controlling YAP1/TAZ at both the transcript and protein levels. Kindlin-2 physically associates with myosin light-chain kinase in response to mechanical cues of cell microenvironment and intracellular signaling events and promotes myosin light-chain phosphorylation. Loss of kindlin-2 inhibits RhoA activation and reduces myosin light-chain phosphorylation, stress fiber formation, and focal adhesion assembly, resulting in increased Ser127 phosphorylation, nuclear exclusion, and ubiquitin ligase atrophin-1 interacting protein 4-mediated degradation of YAP1/TAZ. Our findings reveal a novel kindlin-2 signaling axis that senses the mechanical cues of cell microenvironment and controls MSC fate decision, and they suggest a new strategy to regulate MSC differentiation, tissue repair, and regeneration.

Maslinic acid promotes autophagy by disrupting the interaction between Bcl2 and Beclin1 in rat pheochromocytoma PC12 cells.

Maslinic acid (2α, 3ß-dihydroxyolean-12-en-28-oic acid, MA) was isolated from natural plants and showed anti-cancer activity in rat Pheochromocytoma PC12 cells in our previous studies. We now discover that MA disrupts the interaction between Bcl2 and autophagy scaffold protein Beclin1 in the above cell line, leading to the up-regulation of autophagy. We investigated the effect of MA on the interaction between Bcl2 and Beclin1 by biochemical and biophysical methods in combination with autophagy characterization in the above cell line. Our results suggest that MA may serve as an autophagy activator by directly blocking the Bcl2-Beclin1 interaction to release free Beclin1 required for the recruitment of autophagy positive regulators, implying MA may exert its anti-cancer activity by regulating autophagy.