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 enhances c-Myc translation through association with DDX3X to promote pancreatic ductal adenocarcinoma progression.

Rationale: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid tumor, with extremely low survival rates. Identifying key signaling pathways driving PDAC progression is crucial for the development of therapies to improve patient response rates. Kindlin-2, a multi-functional protein, is involved in numerous biological processes including cell proliferation, apoptosis and migration. However, little is known about the functions of Kindlin-2 in pancreatic cancer progression in vivo. Methods: In this study, we employ an in vivo PDAC mouse model to directly investigate the role of Kindlin-2 in PDAC progression. Then, we utilized RNA-sequencing, the molecular and cellular assays to determine the molecular mechanisms by which Kindlin-2 promotes PDAC progression. Results: We show that loss of Kindlin-2 markedly inhibits KrasG12D-driven pancreatic cancer progression in vivo as well as in vitro. Furthermore, we provide new mechanistic insight into how Kindlin-2 functions in this process, A fraction of Kindlin-2 was localized to the endoplasmic reticulum and associated with the RNA helicase DDX3X, a key regulator of mRNA translation. Loss of Kindlin-2 blocked DDX3X from binding to the 5'-untranslated region of c-Myc and inhibited DDX3X-mediated c-Myc translation, leading to reduced c-Myc-mediated glucose metabolism and tumor growth. Importantly, restoration of the expression of either the full-length Kindlin-2 or c-Myc, but not that of a DDX3X-binding-defective mutant of Kindlin-2, in Kindlin-2 deficient PDAC cells, reversed the inhibition of glycolysis and pancreatic cancer progression induced by the loss of Kindlin-2. Conclusion: Our studies reveal a novel Kindlin-2-DDX3X-c-Myc signaling axis in PDAC progression and suggest that inhibition of this signaling axis may provide a promising therapeutic approach to alleviate PDAC progression.

CHILKBP protects against podocyte injury by preserving ZO-1 expression.

Glomerular diseases afflict millions of people and impose an enormous burden on public healthcare costs worldwide. Identification of potential therapeutic targets for preventing glomerular diseases is of considerable clinical importance. CHILKBP is a focal adhesion protein and modulates a wide array of biological functions. However, little is known about the role of CHILKBP in glomerular diseases. To investigate the function of CHILKBP in maintaining the structure and function of podocytes in a physiologic setting, a mouse model (CHILKBP cKO) was generated in which CHILKBP gene was conditionally deleted in podocytes using the Cre-LoxP system. Ablation of CHILKBP in podocytes resulted in massive proteinuria and kidney failure in mice. Histologically, typical podocyte injury including podocyte loss, foot process effacement, and glomerulosclerosis was observed in CHILKBP cKO mice. Mechanistically, we identified ZO-1 as a key junctional protein that interacted with CHILKBP. Loss of CHILKBP in podocytes exhibited a significant reduction of ZO-1 expression, leading to abnormal actin organization, aberrant slit diaphragm protein expression and compromised podocyte filtration capacity. Restoration of CHILKBP or ZO-1 in CHILKBP-deficient podocytes effectively alleviated podocyte injury induced by the loss of CHILKBP in vitro and in vivo. Finally, we showed the glomerular expression of CHILKBP and ZO-1 was decreased in patients with proteinuric kidney diseases. Our findings reveal a novel signaling pathway consisting of CHILKBP and ZO-1 that plays an essential role in maintaining podocyte homeostasis and suggest novel therapeutic approaches to alleviate glomerular diseases.

CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1.

Background: p53 deficiency is a key causal factor for tumor development and progression. p53 acts in this process through, at least in part, cooperation with YAP1 but the underlying molecular mechanism is incompletely understood. In this paper, we show that CLP36, an actinin-binding cytoskeletal protein, links p53 deficiency to up-regulation of YAP1 expression and sarcoma progression. Methods: Immunohistochemical staining and Western blotting were used to investigate the effect of p53 deficiency on CLP36 expression in sarcoma tissues and cells. Furthermore, molecular, cellular, and genetic knockout and knockdown approaches were employed to investigate the functions of CLP36 in regulation of sarcoma cell behavior in culture and tumor growth in mice. Finally, biochemical approaches were used to investigate the molecular mechanism by which CLP36 regulates the malignant behavior of p53 deficient sarcoma cells. Results: We have found that the expression of CLP36 is up-regulated in response to loss of p53 in sarcoma tissues and cells. Depletion of CLP36 inhibited malignant behavior of p53 deficient sarcoma cells. Furthermore, knockout of CLP36 in mice markedly inhibited p53 deficiency-induced tumorigenesis and improved the survival of the p53 deficient mice. Mechanistically, CLP36 promoted p53 deficiency-induced tumorigenesis through inhibition of E3 ligase atrophin-1 interacting protein-4 (AIP-4)-dependent proteasomal degradation of YAP1 and consequently increase of YAP1 expression. Conclusions: Our results reveal a crucial role of CLP36 in linking p53 deficiency to up-regulation of YAP1 expression and sarcoma progression. Our findings suggest that therapeutic targeting the CLP36/YAP1 signaling axis may provide an effective strategy for alleviation of p53 deficient sarcoma progression.

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression.

Androgen receptor (AR) signaling plays important roles in breast cancer progression. We show here that Kindlin-2, a focal adhesion protein, is critically involved in the promotion of AR signaling and breast cancer progression. Kindlin-2 physically associates with AR and Src through its two neighboring domains, namely F1 and F0 domains, resulting in formation of a Kindlin-2-AR-Src supramolecular complex and consequently facilitating Src-mediated AR Tyr-534 phosphorylation and signaling. Depletion of Kindlin-2 was sufficient to suppress Src-mediated AR Tyr-534 phosphorylation and signaling, resulting in diminished breast cancer cell proliferation and migration. Re-expression of wild-type Kindlin-2, but not AR-binding-defective or Src-binding-defective mutant forms of Kindlin-2, in Kindlin-2-deficient cells restored AR Tyr-534 phosphorylation, signaling, breast cancer cell proliferation and migration. Furthermore, re-introduction of phosphor-mimic mutant AR-Y534D, but not wild-type AR reversed Kindlin-2 deficiency-induced inhibition of AR signaling and breast cancer progression. Finally, using a genetic knockout strategy, we show that ablation of Kindlin-2 from mammary tumors in mouse significantly reduced AR Tyr-534 phosphorylation, breast tumor progression and metastasis in vivo. Our results suggest a critical role of Kindlin-2 in promoting breast cancer progression and shed light on the molecular mechanism through which it functions in this process.

PINCH-1 promotes IGF-1 receptor expression and skin cancer progression through inhibition of the GRB10-NEDD4 complex.

Background: Insulin-like growth factor 1 receptor (IGF-1R) expression and signaling play important roles in promotion of skin cancer progression. Identification of signaling pathways that regulate IGF-1R is crucial for understanding the pathogenesis and therapeutic treatment of skin cancer. Methods: Molecular, cellular and genetic approaches were used to investigate the function of PINCH-1 in regulation of IGF-1R expression and skin cell behavior. Furthermore, conditional PINCH-1 knockout mouse and carcinogen (7, 12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA))-induced skin cancer model were employed to determine the function of PINCH-1 in regulation of IGF-1R expression and skin carcinogenesis in vivo. Results: Knockdown of PINCH-1 from HaCaT keratinocytes or A431 squamous carcinoma cells diminished IGF-1R levels, suppressed cell proliferation and increased apoptosis. Re-expression of PINCH-1 in PINCH-1 knockdown cells restored IGF-1R expression, cell proliferation and survival. Furthermore, depletion of NEDD4 effectively reversed PINCH-1 deficiency-induced down-regulation of IGF-1R expression, cell proliferation and survival. Conditional knockout of PINCH-1 from keratin 5 (K5) positive keratinocytes in mice, like depletion of PINCH-1 from keratinocytes in culture, reduced the IGF-1R level. Using a mouse model of DMBA/TPA-induced skin cancer, we show that the levels of both PINCH-1 and IGF-1R were significantly increased in response to treatment with the carcinogens. Genetic ablation of PINCH-1 from the epidermis markedly reduced the IGF-1R expression and cell proliferation despite stimulation with DMBA/TPA, resulting in resistance to chemical carcinogen-induced skin cancer initiation and progression. Conclusions: Our results reveal a PINCH-1-NEDD4-IGF-1R signaling axis that is critical for promotion of skin tumorigenesis and suggest a new strategy for therapeutic control of skin cancer progression.

Akt isoforms differentially provide for chemoresistance in prostate cancer.

OBJECTIVE: Early prostate cancer micrometastatic foci undergo a mesenchymal to epithelial reverting transition, not only aiding seeding and colonization, but also rendering the tumor cells generally chemoresistant. We previously found that upregulated E-cadherin in the epithelial micrometastases activated canonical survival pathways, including PI3K-Akt, that protected the tumor cells from death; however, the extent of protection from blocking the pathway in its entirety was modest, because different isoforms may have alternately affected cell functioning. Here, we characterized Akt isoform expressions in primary and metastatic prostate cancers, as well as their individual contributions to chemoresistance. METHODS: Akt isoforms and E-cadherin were manipulated with drugs, knocked down, and over expressed. Tumor cell killing was determined in vitro and in vivo. Overall survival was calculated from patient records and specimens. RESULTS: Pan-Akt inhibition sensitized tumor cells to chemotherapy, and specific blockade of Akt1 or/and Akt2 caused cells to be more chemoresponsive. Overexpression of Akt3 induced apoptosis. A low dose of Akt1 or Akt2 inhibitor enabled standard chemotherapies to significantly eradicate metastatic prostate tumors in a mouse model, acting as chemosensitizers. In human specimens, we found Akt1 and Akt2 positively correlated, whereas Akt3 inversely correlated, with the overall survival of prostate cancer patients. Akt1high/Akt2high/Akt3low tumors had the worst outcomes. CONCLUSIONS: E-cadherin-induced activation of Akt1/2 isoforms was the essential mechanism of chemoresistance, whereas Akt3 made cells more fragile. These findings emphasized the need to target Akt1/2, rather than pan-Akt, as a rational therapeutic approach.

How signaling pathways link extracellular mechano-environment to proline biosynthesis: A hypothesis: PINCH-1 and kindlin-2 sense mechanical signals from extracellular matrix and link them to proline biosynthesis.

We propose a signaling pathway in which cell-extracellular matrix (ECM) adhesion components PINCH-1 and kindlin-2 sense mechanical signals from ECM and link them to proline biosynthesis, a vital metabolic pathway for macromolecule synthesis, redox balance, and ECM remodeling. ECM stiffening promotes PINCH-1 expression via integrin signaling, which suppresses dynamin-related protein 1 (DRP1) expression and mitochondrial fission, resulting in increased kindlin-2 translocation into mitochondria and interaction with Δ1 -pyrroline-5-carboxylate (P5C) reductase 1 (PYCR1). Kindlin-2 interaction with PYCR1 protects the latter from proteolytic degradation, leading to elevated PYCR1 level. Additionally, PINCH-1 promotes P5C synthase (P5CS) expression and P5C synthesis, which, together with increased PYCR1 level, support augmented proline biosynthesis. This signaling pathway is frequently activated in fibrosis and cancer, resulting in increased proline biosynthesis and excessive collagen matrix production, which in turn further promotes ECM stiffening. Targeting this signaling pathway, therefore, may provide an effective strategy for alleviating fibrosis and cancer progression.

PINCH-1 promotes Δ1-pyrroline-5-carboxylate synthase expression and contributes to proline metabolic reprogramming in lung adenocarcinoma.

Proline metabolic reprogramming is intimately involved in cancer progression. We recently identified a critical role of PINCH-1, a cell-extracellular matrix (ECM) adhesion protein whose expression is elevated in lung adenocarcinoma, in the promotion of proline biosynthesis, fibrosis and lung adenocarcinoma growth. How PINCH-1 promotes proline biosynthesis, however, was incompletely understood. In this study, we show that PINCH-1 promotes the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), a key enzyme that links glutamate metabolism to proline biosynthesis. Depletion of PINCH-1 from lung adenocarcinoma cells reduced the protein but not mRNA level of P5CS, resulting in down-regulation of the cellular level of P5C and cell proliferation. Treatment of the cells with protease inhibitor leupeptin effectively reversed PINCH-1 deficiency-induced reduction of the P5CS level. At the molecular level, PINCH-1, through its LIM2 domain, physically associated with P5CS in lung adenocarcinoma cells. Re-expression of wild type PINCH-1, but not that of the PINCH-1 LIM2 deletion mutant, in PINCH-1 deficient lung adenocarcinoma cells restored P5CS expression, proline biosynthesis and cell proliferation. Finally, P5CS expression, like that of PINCH-1, is elevated in human and mouse lung adenocarcinoma. Using a mouse model of lung adenocarcinoma in which PINCH-1 is conditionally ablated, we show that knockout of PINCH-1 from lung adenocarcinoma effectively reduced the P5CS level in vivo. Our results reveal an important role of PINCH-1 in the promotion of P5CS expression, which likely contributes to proline metabolic reprogramming and consequently lung adenocarcinoma progression.

RSU-1 interaction with prohibitin-2 links cell-extracellular matrix detachment to downregulation of ERK signaling.

Cell-extracellular matrix (ECM) detachment is known to decrease extracellular signal-regulated kinase (ERK) signaling, an intracellular pathway that is central for control of cell behavior. How cell-ECM detachment is linked to downregulation of ERK signaling, however, is incompletely understood. We show here that focal adhesion protein Ras Suppressor 1 (RSU1) plays a critical role in cell-ECM detachment induced suppression of ERK signaling. We have identified prohibitin 2 (PHB2), a component of membrane lipid rafts, as a novel binding protein of RSU1, and mapped a major RSU1-binding site to PHB2 amino acids 150 to 206 in the C-terminal region of the PHB/SPFH (stomatin/prohibitin/flotillin/HflKC) domain. The PHB2 binding is mediated by multiple sites located in the N-terminal leucine-rich repeat region of RSU1. Depletion of PHB2 suppressed cell-ECM adhesion-induced ERK activation. Furthermore, cell-ECM detachment increased RSU1 association with membrane lipid rafts and interaction with PHB2. Finally, knockout of RSU1 or inhibition of RSU1 interaction with PHB2 by overexpression of the major RSU1-binding PHB2 fragment (amino acids 150-206) effectively suppressed the cell-ECM detachment induced downregulation of ERK signaling. Additionally, expression of venus-tagged wild-type RSU1 restored ERK signaling, while expression of venus-tagged PHB2-binding defective RSU1 mutant in which the N-terminal leucine-rich repeat region is deleted did not. Taken together, Our findings identify a novel RSU1-PHB2 signaling axis that senses cell-ECM detachment and links it to decreased ERK signaling.

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.

A mechanoresponsive PINCH-1-Notch2 interaction regulates smooth muscle differentiation of human placental mesenchymal stem cells.

Extracellular matrix (ECM) stiffness plays an important role in the decision making process of smooth muscle differentiation of mesenchymal stem cells (MSCs) but the underlying mechanisms are incompletely understood. Here we show that a signaling axis consisting of PINCH-1 and Notch2 is critically involved in mediating the effect of ECM stiffness on smooth muscle differentiation of MSCs. Notch2 level is markedly increased in ECM stiffness-induced smooth muscle differentiation of human placental MSCs. Knockdown of Notch2 from human placental MSCs effectively inhibits ECM stiffness-induced smooth muscle differentiation, whereas overexpression of North intracellular domain (NICD2) is sufficient to drive human placental MSC differentiation toward smooth muscle cells. At the molecular level, Notch2 directly interacts with PINCH-1. The interaction of Notch2 with PINCH-1 is significantly increased in response to ECM stiffness favoring smooth muscle differentiation. Furthermore, depletion of PINCH-1 from human placental MSCs reduces Notch2 level and consequently suppresses ECM stiffness-induced smooth muscle differentiation. Re-expression of PINCH-1, but not that of a Notch2-binding defective PINCH-1 mutant, in PINCH-1 knockdown human placental MSCs restores smooth muscle differentiation. Finally, overexpression of NICD2 is sufficient to override PINCH-1 deficiency-induced defect in smooth muscle differentiation. Our results identify an ECM stiffness-responsive PINCH-1-Notch2 interaction that is critically involved in ECM stiffness-induced smooth muscle differentiation of human placental MSCs.

Complex structures of Rsu1 and PINCH1 reveal a regulatory mechanism of the ILK/PINCH/Parvin complex for F-actin dynamics.

Communications between actin filaments and integrin-mediated focal adhesion (FA) are crucial for cell adhesion and migration. As a core platform to organize FA proteins, the tripartite ILK/PINCH/Parvin (IPP) complex interacts with actin filaments to regulate the cytoskeleton-FA crosstalk. Rsu1, a Ras suppressor, is enriched in FA through PINCH1 and plays important roles in regulating F-actin structures. Here, we solved crystal structures of the Rsu1/PINCH1 complex, in which the leucine-rich-repeats of Rsu1 form a solenoid structure to tightly associate with the C-terminal region of PINCH1. Further structural analysis uncovered that the interaction between Rsu1 and PINCH1 blocks the IPP-mediated F-actin bundling by disrupting the binding of PINCH1 to actin. Consistently, overexpressing Rsu1 in HeLa cells impairs stress fiber formation and cell spreading. Together, our findings demonstrated that Rsu1 is critical for tuning the communication between F-actin and FA by interacting with the IPP complex and negatively modulating the F-actin bundling.

Kindlin-2 promotes rear focal adhesion disassembly and directional persistence during cell migration.

Cell migration involves front-to-rear asymmetric focal adhesion (FA) dynamics, which facilitates trailing edge detachment and directional persistence. Here, we show that kindlin-2 is crucial for FA sliding and disassembly in migrating cells. Loss of kindlin-2 markedly reduced FA number and selectively impaired rear FA sliding and disassembly, resulting in defective rear retraction and reduced directional persistence during cell migration. Kindlin-2-deficient cells failed to develop serum-induced actomyosin-dependent tension at FAs. At the molecular level, kindlin-2 directly interacted with myosin light chain kinase (MYLK, hereafter referred to as MLCK), which was enhanced in response to serum stimulation. Serum deprivation inhibited rear FA disassembly, which was released in response to serum stimulation. Overexpression of the MLCK-binding kindlin-2 F0F1 fragment (amino acid residues 1-167), which inhibits the interaction of endogenous kindlin-2 with MLCK, phenocopied kindlin-2 deficiency-induced migration defects. Inhibition of MLCK, like loss of kindlin-2, also impaired trailing-edge detachment, rear FA disassembly and directional persistence. These results suggest a role of kindlin-2 in promoting actomyosin contractility at FAs, leading to increased rear FA sliding and disassembly, and directional persistence during cell migration.

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.

RSU-1 Maintains Integrity of Caenorhabditis elegans Vulval Muscles by Regulating α-Actinin.

Egg-laying behavior in Caenorhabditis elegans is a well-known model for investigating fundamental cellular processes. In egg-laying, muscle contraction is the relaxation of the vulval muscle to extrude eggs from the vulva. Unlike skeletal muscle, vulval muscle lacks visible striations of the sarcomere. Therefore, vulval muscle must counteract the mechanical stress, caused by egg extrusion and body movement, from inducing cell-shape distortion by maintaining its cytoskeletal integrity. However, the underlying mechanisms that regulate the cellular integrity in vulval muscles remain unclear. Here, we demonstrate that C. elegans egg-laying requires proper vulval muscle 1 (vm1), in which the actin bundle organization of vm1 muscles is regulated by Ras suppressor protein 1 (RSU-1). In the loss of RSU-1, as well as RasLET-60 overactivation, blister-like membrane protrusions and disorganized actin bundles were observed in the vm1 muscles. Moreover, RasLET-60 depletion diminished the defected actin-bundles in rsu-1 mutant. These results reveal the genetic interaction of RSU-1 and RasLET-60in vivo In addition, our results further demonstrated that the fifth to seventh leucine-rich region of RSU-1 is required to promote actin-bundling protein, α-actinin, for actin bundle stabilization in the vm1 muscles. This expands our understanding of the molecular mechanisms of actin bundle organization in a specialized smooth muscle.

Focal adhesion protein Kindlin-2 regulates bone homeostasis in mice.

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes, downregulates ß-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation of ß-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKL-neutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation. Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.

Kindlin-2 modulates MafA and ß-catenin expression to regulate ß-cell function and mass in mice.

ß-Cell dysfunction and reduction in ß-cell mass are hallmark events of diabetes mellitus. Here we show that ß-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in ß-cells. Kindlin-2 loss activates GSK-3ß and downregulates ß-catenin, leading to reduced ß-cell proliferation and mass. Kindlin-2 loss reduces the percentage of ß-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of ß-catenin in ß-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of ß-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes.

Mitochondrial dynamics links PINCH-1 signaling to proline metabolic reprogramming and tumor growth.

Proline metabolism is critical for cellular response to microenvironmental stress in living organisms across different kingdoms, ranging from bacteria, plants to animals. In bacteria and plants, proline is known to accrue in response to osmotic and other stresses. In higher organisms such as human, proline metabolism plays important roles in physiology as well as pathological processes including cancer. The importance of proline metabolism in physiology and diseases lies in the fact that the products of proline metabolism are intimately involved in essential cellular processes including protein synthesis, energy production and redox signaling. A surge of protein synthesis in fast proliferating cancer cells, for example, results in markedly increased demand for proline. Proline synthesis is frequently unable to meet the demand in fast proliferating cancer cells. The inadequacy of proline or "proline vulnerability" in cancer may provide an opportunity for therapeutic control of cancer progression. To this end, it is important to understand the signaling mechanism through which proline synthesis is regulated. In a recent study (Guo et al., Nat Commun 11(1):4913, doi: 10.1038/s41467-020-18753-6), we have identified PINCH-1, a component of cell-extracellular matrix (ECM) adhesions, as an important regulator of proline synthesis and cancer progression.

PINCH-1 interacts with myoferlin to promote breast cancer progression and metastasis.

PINCH-1 is a cytoplasmic component of the cell-extracellular matrix (ECM) adhesion machine that is frequently overexpressed in cancer. The functions and mechanism of PINCH-1 in cancer, however, remain to be determined. Here, we show that PINCH-1 interacts with myoferlin, a transmembrane protein that is critical for cancer progression. High expression of both PINCH-1 and myoferlin correlates with poor clinical outcome in human breast cancer patients. Ablation of PINCH-1 from breast cancer cells diminished myoferlin level and suppressed breast cancer cell proliferation, migration, and endothelial cell tube formation in vitro and breast tumor growth, angiogenesis and metastasis in vivo. Mechanistically, PINCH-1 controls myoferlin level through its interaction with myoferlin and regulation of its ubiquitination and proteasome-dependent degradation. Functionally, re-expression of PINCH-1, but not that of a myoferlin-binding defectiveΔLIM2 mutant, effectively reversed the inhibition of myoferlin expression and breast cancer progression induced by loss of PINCH-1. Finally, restoration of myoferlin expression was sufficient to reverse PINCH-1-deficiency induced inhibition on breast cancer progression. These results reveal a PINCH-1-myoferlin signaling axis that is critical for breast cancer progression and suggest a new strategy for therapeutic control of breast cancer.