ANGPTL4 binds to the leptin receptor to regulate ectopic bone formation.

Leptin protein was thought to be unique to leptin receptor (LepR), but the phenotypes of mice with mutation in LepR [db/db (diabetes)] and leptin [ob/ob (obese)] are not identical, and the cause remains unclear. Here, we show that db/db, but not ob/ob, mice had defect in tenotomy-induced heterotopic ossification (HO), implicating alternative ligand(s) for LepR might be involved. Ligand screening revealed that ANGPTL4 (angiopoietin-like protein 4), a stress and fasting-induced factor, was elicited from brown adipose tissue after tenotomy, bound to LepR on PRRX1+ mesenchymal cells at the HO site, thus promotes chondrogenesis and HO development. Disruption of LepR in PRRX1+ cells, or lineage ablation of LepR+ cells, or deletion of ANGPTL4 impeded chondrogenesis and HO in mice. Together, these findings identify ANGPTL4 as a ligand for LepR to regulate the formation of acquired HO.

TREM2 acts as a receptor for IL-34 to suppress acute myeloid leukemia in mice.

The bone marrow microenvironment supports leukocyte mobilization and differentiation and controls the development of leukemias, including acute myeloid leukemia (AML). Here, we found that the development of AML xenotransplants was suppressed in mice with osteoclasts tuberous sclerosis 1 (Tsc1) deletion. Tsc1-deficient osteoclasts released a high level of interleukin-34 (IL-34), which efficiently induced AML cell differentiation and prevented AML progression in various preclinical models. Conversely, AML development was accelerated in mice deficient in IL-34. Interestingly, IL-34 inhibited AML independent of its known receptors but bound directly to triggering receptor expressed on myeloid cells 2 (TREM2), a key hub of immune signals. TREM2-deficient AML cells and normal myeloid cells were resistant to IL-34 treatment. Mechanistically, IL-34-TREM2 binding rapidly phosphorylated Ras protein activator like 3 and inactivated extracellular signal-regulated protein kinase 1/2 signaling to prevent AML cell proliferation and stimulate differentiation. Furthermore, TREM2 was downregulated in patients with AML and associated with a poor prognosis. This study identified TREM2 as a novel receptor for IL-34, indicating a promising strategy for overcoming AML differentiation blockade in patients with AML.

Rheb1 is required for limb growth through regulating chondrogenesis in growth plate.

Ras homology enriched in the brain (Rheb) is well established as a critical regulator of cell proliferation and differentiation in response to growth factors and nutrients. However, the role of Rheb1 in limb development remains unknown. Here, we found that Rheb1 was dynamically expressed during the proliferation and differentiation of chondrocytes in the growth plate. Given that Prrx1+ limb-bud-like mesenchymal cells are the source of limb chondrocytes and are essential for endochondral ossification, we conditionally deleted Rheb1 using Prrx1-Cre and found a limb dwarfism in Prrx1-Cre; Rheb1fl/fl mice. Normalized to growth plate height, the conditional knockout (cKO) mice exhibited a significant decrease in column count of proliferative zones which was increased in hypertrophic zones resulting in decreased growth plate size, indicating abnormal endochondral ossification. Interestingly, although Rheb1 deletion profoundly inhibited the transcription factor Sox9 in limb cartilage; levels of runx2 and collagen type 2 were both increased. These novel findings highlight the essential role of Rheb1 in limb growth and indicate a complex regulation of Rheb1 in chondrocyte proliferation and differentiation.

Tsc1 regulates tight junction independent of mTORC1.

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor that functions together with Tsc2 to negatively regulate the mechanistic target of rapamycin complex 1 (mTORC1) activity. Here, we show that Tsc1 has a critical role in the tight junction (TJ) formation of epithelium, independent of its role in Tsc2 and mTORC1 regulation. When an epithelial cell establishes contact with neighboring cells, Tsc1, but not Tsc2, migrates from the cytoplasm to junctional membranes, in which it binds myosin 6 to anchor the perijunctional actin cytoskeleton to ß-catenin and ZO-1. In its absence, perijunctional actin cytoskeleton fails to form. In mice, intestine-specific or inducible, whole-body Tsc1 ablation disrupts adherens junction/TJ structures in intestine or skin epithelia, respectively, causing Crohn's disease-like symptoms in the intestine or psoriasis-like phenotypes on the skin. In patients with Crohn's disease or psoriasis, junctional Tsc1 levels in epithelial tissues are markedly reduced, concomitant with the TJ structure impairment, suggesting that Tsc1 deficiency may underlie TJ-related diseases. These findings establish an essential role of Tsc1 in the formation of cell junctions and underpin its association with TJ-related human diseases.

Establishment of bovine expanded potential stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Abemaciclib drives the therapeutic differentiation of acute myeloid leukaemia stem cells.

Self-renewal and differentiation arrest are two features of leukaemia stem cells (LSCs) responsible for the high relapse rate of acute myeloid leukaemia (AML). To screen drugs to overcome differentiation blockade for AML, we conducted screening of 2040 small molecules from a library of United States Food and Drug Administration-approved drugs and found that the cyclin-dependent kinase (CDK)4/6 inhibitor, abemaciclib, exerts high anti-leukaemic activity. Abemaciclib significantly suppressed proliferation and promoted the differentiation of LSCs in vitro. Abemaciclib also efficiently induced differentiation and impaired self-renewal of LSCs, thus reducing the leukaemic cell burden and improving survival in various preclinical animal models, including patient-derived xenografts. Importantly, abemaciclib strongly enhanced anti-tumour effects in combination with venetoclax, a B-cell lymphoma 2 (Bcl-2) inhibitor. This treatment combination led to a marked decrease in LSC-enriched populations and resulted in a synergistic anti-leukaemic effect. Target screening revealed that in addition to CDK4/6, abemaciclib bound to and inhibited CDK9, consequently attenuating the protein levels of global p-Ser2 RNA Polymerase II (Pol II) carboxy terminal domain (CTD), Myc, Bcl-2, and myeloid cell leukaemia-1 (Mcl-1), which was important for the anti-AML effect of abemaciclib. Collectively, these data provide a strong rationale for the clinical evaluation of abemaciclib to induce LSC differentiation and treat highly aggressive AML as well as other advanced haematological malignancies.

Metformin attenuates osteoarthritis by targeting chondrocytes, synovial macrophages and adipocytes.

OBJECTIVE: To investigate the therapeutic effect and mechanism of metformin on knee OA in normal diet (ND) mice or high-fat diet (HFD)-induced obese mice. METHODS: Destabilization of the medial meniscus surgery was performed in ND mice or HFD mice, and metformin was administrated in drinking water or not. The changes of OA joint structure, infiltration and polarization of synovial macrophages and circulating and local levels of leptin and adiponectin were evaluated. In vitro, the effects of metformin on chondrocytes and macrophages, and of conditioned mediums derived from mouse abdominal fat on murine chondrogenic cell line ATDC5 and murine macrophage cell line RAW264.7, were detected. RESULTS: Metformin showed protective effects on OA, characterized by reductions on OARSI score [2.00, 95% CI (1.15, 2.86) for ND mice and 3.17, 95% CI (2.37, 3.96) for HFD mice] and synovitis score [1.17, 95% CI (0.27, 2.06) for ND mice and 2.50, 95% CI (1.49, 3.51) for HFD mice] after 10 weeks of treatment, and the effects were more significant in HFD mice than in ND mice. Mechanistically, in addition to decreasing apoptosis and matrix-degrading enzymes expression in chondrocytes as well as infiltration and pro-inflammatory differentiation of synovial macrophages, metformin reduced leptin secretion by adipose tissue in HFD mice. CONCLUSIONS: Metformin protects against knee OA which could be through reducing apoptosis and catabolism of chondrocytes, and suppressing infiltration and pro-inflammatory polarization of synovial macrophages. For obese mice, metformin has a greater protective effect in knee OA additionally through reducing leptin secretion from adipose tissue.

Osteocytes regulate neutrophil development through IL-19: a potent cytokine for neutropenia treatment.

Osteocytes are the most abundant (90% to 95%) cells in bone and have emerged as an important regulator of hematopoiesis, but their role in neutrophil development and the underlying mechanisms remain unclear. Interleukin 19 (IL-19) produced predominantly by osteocytes stimulated granulopoiesis and neutrophil formation, which stimulated IL-19 receptor (IL-20Rß)/Stat3 signaling in neutrophil progenitors to promote their expansion and neutrophil formation. Mice with constitutive activation of mechanistic target of rapamycin complex (mTORC1) signaling in osteocytes (Dmp1-Cre) exhibited a dramatic increase in IL-19 production and promyelocyte/myelocytic expansion, whereas mTORC1 inactivation in osteocytes reduced IL-19 production and neutrophil numbers in mice. We showed that IL-19 administration stimulated neutrophil development, whereas neutralizing endogenous IL-19 or depletion of its receptor inhibited the process. Importantly, low-dose IL-19 reversed chemotherapy, irradiation, or chloramphenicol-induced neutropenia in mice more efficiently than granulocyte colony-stimulating factor. This evidence indicated that IL-19 was an essential regulator of neutrophil development and a potent cytokine for neutropenia treatment.

Exploring the translational potential of PLGA nanoparticles for intra-articular rapamycin delivery in osteoarthritis therapy.

Osteoarthritis (OA) is a prevalent joint disease that affects all the tissues within the joint and currently lacks disease-modifying treatments in clinical practice. Despite the potential of rapamycin for OA disease alleviation, its clinical application is hindered by the challenge of achieving therapeutic concentrations, which necessitates multiple injections per week. To address this issue, rapamycin was loaded into poly(lactic-co-glycolic acid) nanoparticles (RNPs), which are nontoxic, have a high encapsulation efficiency and exhibit sustained release properties for OA treatment. The RNPs were found to promote chondrogenic differentiation of ATDC5 cells and prevent senescence caused by oxidative stress in primary mouse articular chondrocytes. Moreover, RNPs were capable to alleviate metabolism homeostatic imbalance of primary mouse articular chondrocytes in both monolayer and 3D cultures under inflammatory or oxidative stress. In the mouse destabilization of the medial meniscus (DMM) model, intra-articular injection of RNPs effectively mitigated joint cartilage destruction, osteophyte formation, chondrocytes hypertrophy, synovial inflammation, and pain. Our study demonstrates the feasibility of using RNPs as a potential clinically translational therapy to prevent the progression of post-traumatic OA.

Excess dietary zinc drives a Cushing's-like syndrome in ovariectomized mice - Implications for postmenopausal obesity.

Postmenopausal women have an increased risk of obesity, but the underlying cause is not clear. We unexpectedly found that excess dietary zinc induced severe obesity and a Cushing's-like syndrome without increased food intake in ovariectomized (Ovx) but not in sham-operated mice. Zinc accumulated in the adrenal glands and inhibited adrenal 17,20-lyase activity and steroid synthesis. As adrenal steroids are the only source of estrogen in Ovx mice, estrogen deficiency induced adrenal hyperplasia, glucocorticoid overproduction, and consequent development of a Cushing's-like syndrome. Adrenal steroid supplementation prevented the effects of zinc. Plasma zinc was positively correlated with cortisol level and negatively correlated with the levels of adrenal steroids and estrogen in obese postmenopausal women. The finding of a link between dietary zinc, estrogen deficiency, and postmenopausal obesity, implies that postmenopausal obesity might be prevented by supplementation with a adrenal steroid and avoiding excess dietary zinc.

Development of Biodegradable Osteopromotive Citrate-Based Bone Putty.

The burden of bone fractures demands development of effective biomaterial solutions, while additional acute events such as noncompressible bleeding further motivate the search for multi-functional implants to avoid complications including osseous hemorrhage, infection, and nonunion. Bone wax has been widely used in orthopedic bleeding control due to its simplicity of use and conformation to irregular defects; however, its nondegradability results in impaired bone healing, risk of infection, and significant inflammatory responses. Herein, a class of intrinsically fluorescent, osteopromotive citrate-based polymer/hydroxyapatite (HA) composites (BPLP-Ser/HA) as a highly malleable press-fit putty is designed. BPLP-Ser/HA putty displays mechanics replicating early nonmineralized bone (initial moduli from ≈2-500 kPa), hydration induced mechanical strengthening in physiological conditions, tunable degradation rates (over 2 months), low swelling ratios (<10%), clotting and hemostatic sealing potential (resistant to blood pressure for >24 h) and significant adhesion to bone (≈350-550 kPa). Simultaneously, citrate's bioactive properties result in antimicrobial (≈100% and 55% inhibition of S. aureus and E. coli) and osteopromotive effects. Finally, BPLP-Ser/HA putty demonstrates in vivo regeneration in a critical-sized rat calvaria model equivalent to gold standard autograft. BPLP-Ser/HA putty represents a simple, off-the-shelf solution to the combined challenges of acute wound management and subsequent bone regeneration.

Loss of Raptor induces Sertoli cells into an undifferentiated state in mice.

In mammals, testis development is triggered by the expression of the sex-determining Y-chromosome gene SRY to commit the Sertoli cell (SC) fate at gonadal sex determination in the fetus. Several genes have been identified to be required to promote the testis pathway following SRY activation (i.e., SRY box 9 (SOX9)) in an embryo; however, it largely remains unknown about the genes and the mechanisms involved in stabilizing the testis pathway after birth and throughout adulthood. Herein, we report postnatal males with SC-specific deletion of Raptor demonstrated the absence of SC unique identity and adversely acquired granulosa cell-like characteristics, along with loss of tubular architecture and scattered distribution of SCs and germ cells. Subsequent genome-wide analysis by RNA sequencing revealed a profound decrease in the transcripts of testis genes (i.e., Sox9, Sox8, and anti-Mullerian hormone (Amh)) and, conversely, an increase in ovary genes (i.e., LIM/Homeobox gene 9 (Lhx9), Forkhead box L2 (Foxl2) and Follistatin (Fst)); these changes were further confirmed by immunofluorescence and quantitative reverse-transcription polymerase chain reaction. Importantly, co-immunofluorescence demonstrated that Raptor deficiency induced SCs dedifferentiation into a progenitor state; the Raptor-mutant gonads showed some ovarian somatic cell features, accompanied by enhanced female steroidogenesis and elevated estrogen levels, yet the zona pellucida 3 (ZP3)-positive terminally feminized oocytes were not observed. In vitro experiments with primary SCs suggested that Raptor is likely involved in the fibroblast growth factor 9 (FGF9)-induced formation of cell junctions among SCs. Our results established that Raptor is required to maintain SC identity, stabilize the male pathway, and promote testis development.

Mechanical overloading promotes chondrocyte senescence and osteoarthritis development through downregulating FBXW7.

OBJECTIVES: To investigate the role of mechanical stress in cartilage ageing and identify the mechanistic association during osteoarthritis (OA) progression. METHODS: F-box and WD repeat domain containing 7 (FBXW7) ubiquitin ligase expression and chondrocyte senescence were examined in vitro, in experimental OA mice and in human OA cartilage. Mice with Fbxw7 knockout in chondrocytes were generated and adenovirus-expressing Fbxw7 (AAV-Fbxw7) was injected intra-articularly in mice. Destabilised medial meniscus surgery was performed to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score and the changes in chondrocyte senescence were determined. mRNA sequencing was performed in articular cartilage from Fbxw7 knockout and control mice. RESULTS: Mechanical overloading accelerated senescence in cultured chondrocytes and in mice articular cartilage. FBXW7 was downregulated by mechanical overloading in primary chondrocytes and mice cartilage, and decreased in the cartilage of patients with OA, aged mice and OA mice. FBXW7 deletion in chondrocytes induced chondrocyte senescence and accelerated cartilage catabolism in mice, as manifested by an upregulation of p16INK4A, p21 and Colx and downregulation of Col2a1 and ACAN, which resulted in the exacerbation of OA. By contrast, intra-articular injection of adenovirus expressing Fbxw7 alleviated OA in mice. Mechanistically, mechanical overloading decreased Fbxw7 mRNA transcription and FBXW7-mediated MKK7 degradation, which consequently stimulated JNK signalling. In particular, inhibition of JNK activity by DTP3, a MKK7 inhibitor, ameliorated chondrocyte senescence and cartilage degeneration CONCLUSIONS: FBXW7 is a key factor in the association between mechanical overloading and chondrocyte senescence and cartilage ageing in the pathology of OA.

Asporin regulated by miR-26b-5p mediates chondrocyte senescence and exacerbates osteoarthritis progression via TGF-ß1/Smad2 pathway.

OBJECTIVES: This study aimed to investigate the role and mechanism of asporin in modulating chondrocyte senescence in OA pathology. METHODS: Asporin and senescence-related hallmark expression were examined in human and experimental OA mouse cartilage samples. Twelve-week-old male C57 mice were administered with recombinant protein (rm-asporin)- or asporin-siRNA-expressing lentiviruses via intra-articular injection once a week after destabilization of the medial meniscus (DMM) surgery to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score. Senescence-associated ß-galactosidase (SA-ß-Gal) staining, γH2AX, p21 and p16INK4a were analysed by immunofluorescence staining and western blot to assess the specific role of asporin in chondrocyte senescence. The TGF-ß1-Smad2 signalling pathway and miR-26b-5p were further evaluated to explore the mechanism of asporin in OA. RESULTS: Asporin was upregulated in articular chondrocytes of OA patients and DMM mice and accompanied by accumulation of senescent cells. Asporin overexpression exaggerated OA progression, whereas silencing asporin restored chondrocyte homeostasis and deferred chondrocyte senescence, leading to markedly attenuated DMM-induced OA. Cellular and molecular analyses showed that asporin can be inhibited by miR-26b-5p, which was significantly downregulated in OA cartilage, leading to exacerbation of experimental OA partially through inhibition of TGF-ß1-Smad2 signalling in chondrocytes. CONCLUSIONS: Our findings indicate that asporin plays an essential role in chondrocyte senescence and OA pathogenesis. Upregulated by miR-26b-5p, asporin inhibits the TGF-ß1-Smad2 pathway to accelerate chondrocyte senescence and exacerbate cartilage degeneration. Targeting the miR-26b-5p-asporin-Smad2 axis may serve as a practical therapeutic strategy to delay chondrocyte senescence and OA development.

Osteoarthritic infrapatellar fat pad aggravates cartilage degradation via activation of p38MAPK and ERK1/2 pathways.

OBJECTIVE: This study aimed to investigate the biochemical effects of osteoarthritic infrapatellar fat pad (IPFP) on cartilage and the underlying mechanisms. METHODS: Human IPFP and articular cartilage were collected from end-stage osteoarthritis (OA) patients during total knee arthroplasty. IPFP-derived fat-conditioned medium (FCM) was used to stimulate human primary chondrocytes and cartilage explants. Functional effect of osteoarthritic IPFP was explored in human primary chondrocytes and articular cartilage in vitro and ex vivo. Activation of relative pathways and its effects on chondrocytes were assessed through immunoblotting and inhibition experiments, respectively. Neutralization test was performed to identify the main factors and their associated pathways responsible for the effects of IPFP. RESULTS: Osteoarthritic IPFP-derived FCM significantly induced extracellular matrix (ECM) degradation in both human primary chondrocytes and cartilage explants. Several pathways, such as NF-κB, mTORC1, p38MAPK, JNK, and ERK1/2 signaling, were significantly activated in human chondrocytes with osteoarthritic IPFP-derived FCM stimulation. Interestingly, inhibition of p38MAPK and ERK1/2 signaling pathway could alleviate the detrimental effects of FCM on chondrocytes, while inhibition of other signaling pathways had no similar results. In addition, IL-1ß and TNF-α instead of IL-6 in osteoarthritic IPFP-derived FCM played key roles in cartilage degradation via activating p38MAPK rather than ERK1/2 signaling pathway. CONCLUSION: Osteoarthritic IPFP induces the degradation and inflammation of cartilage via activation of p38MAPK and ERK1/2 pathways, in which IL-1ß and TNF-α act as the key factors. Our study suggests that modulating the effects of IPFP on cartilage may be a promising strategy for knee OA intervention.

Interleukin 9 prevents immune thrombocytopenia in mice via JAK/STAT5 signaling.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoimmune-mediated platelet destruction and impaired platelet production, which can lead to an increased risk of bleeding. The clinical management of ITP currently remains a challenge for hematologists. We explored the role of interleukin-9 (IL-9) in the treatment of CD41-induced ITP, and investigated its underlying mechanisms in a CD41-induced ITP mouse model. IL-9 treatment increased the numbers of mature megakaryocytes (CD41+CD42d+) and CD41+Sca-1+ cells in the bone marrow in these model mice, while IL-9 receptor (IL-9R) small interfering RNA (siRNA) inhibited the process. Moreover, phosphorylated signal transducer and activator of transcription 5 (STAT5), as a downstream molecule of IL-9R, was increased after IL-9 treatment. We next investigated the source of IL-9 in bone marrow, osteoblasts produced the highest level of IL-9. These results confirmed that IL-9 could prevent CD41-induced ITP in BALB/c mice by regulating osteoblasts and activating IL-9R/STAT5 signaling in megakaryocytes, thus providing further evidence for IL-9 as a promising therapeutic agent for the treatment of ITP.

Citrate-based materials fuel human stem cells by metabonegenic regulation.

A comprehensive understanding of the key microenvironmental signals regulating bone regeneration is pivotal for the effective design of bioinspired orthopedic materials. Here, we identified citrate as an osteopromotive factor and revealed its metabonegenic role in mediating citrate metabolism and its downstream effects on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Our studies show that extracellular citrate uptake through solute carrier family 13, member 5 (SLC13a5) supports osteogenic differentiation via regulation of energy-producing metabolic pathways, leading to elevated cell energy status that fuels the high metabolic demands of hMSC osteodifferentiation. We next identified citrate and phosphoserine (PSer) as a synergistic pair in polymeric design, exhibiting concerted action not only in metabonegenic potential for orthopedic regeneration but also in facile reactivity in a fluorescent system for materials tracking and imaging. We designed a citrate/phosphoserine-based photoluminescent biodegradable polymer (BPLP-PSer), which was fabricated into BPLP-PSer/hydroxyapatite composite microparticulate scaffolds that demonstrated significant improvements in bone regeneration and tissue response in rat femoral-condyle and cranial-defect models. We believe that the present study may inspire the development of new generations of biomimetic biomaterials that better recapitulate the metabolic microenvironments of stem cells to meet the dynamic needs of cellular growth, differentiation, and maturation for use in tissue engineering.

ETS2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting JUNB transcription.

Epithelial-to-mesenchymal transition (EMT) plays an important role in the progression of renal tubulointerstitial fibrosis, a common mechanism leading to end-stage renal failure. V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2), a transcription factor, exhibits diverse roles in pathogenesis; however, its role in renal fibrosis is not yet fully understood. In this study, we detected the expression of ETS2 in an animal model of renal fibrosis and evaluated the potential role of ETS2 in tubular EMT induced by TGF-ß1. We found that ETS2 and profibrogenic factors, alpha-smooth muscle actin (α-SMA) and fibronectin (FN), were significantly increased in the unilateral ureteral obstruction (UUO)-induced renal fibrosis model in mice. In vitro, TGF-ß1 induced a high expression of ETS2 dependent on Smad3 and ERK signaling pathway in human proximal tubular epithelial cells (HK2). Knockdown of ETS2 abrogated TGF-ß1-mediated expression of profibrogenic factors vimentin, α-SMA, collagen I, and FN in HK2 cells. Mechanistically, ETS2 promoted JUNB expression in HK2 cells after TGF-ß1 stimulation. Furthermore, luciferase and Chromatin Immunoprecipitation (ChIP) assays revealed that the binding of ETS2 to three EBS motifs on the promoter of JUNB triggered its transcription. Notably, silencing JUNB reversed the ETS2-induced upregulation of the profibrogenic factors in HK2 cells after TGF-ß1 stimulation. These findings suggest that ETS2 mediates TGF-ß1-induced EMT in renal tubular cells through JUNB, a novel pathway for preventing renal fibrosis.

Loss of Fbxw7 in Sertoli cells impairs testis development and causes infertility in mice†.

F-box and WD-40 domain protein 7 (Fbxw7) is a component of the Skp1-Cdc53/Cullin-F-box-protein complex (SCF/ß-TrCP), which is an E3 ubiquitin ligase that mediates protein degradation. This complex has recently been shown to negatively regulate spermatogonial stem cell self-renewal; however, its roles in Sertoli cell (SC) proliferation, differentiation, and function remain to be established. In this study, we generated conditional mutant mice with SC-specific deletion of Fbxw7 via the Cre-loxP system. Fbxw7 deficiency in SCs impaired testis development, which is characterized by age-dependent tubular atrophy, excessive germ cell loss, and spermatogenic arrest, and the mutant males were infertile at 7 months old. Fbxw7 ablation also compromised cytoskeletal organization and cell polarity of SCs, as well as integrity of the blood-testis barrier. In addition, the transcript levels of cell markers for germ cells, Leydig cells, and SCs were significantly decreased in Fbxw7 mutant mice. Importantly, protein levels of GATA-4, a transcription factor that plays a crucial role in SC maturation and testis development, were progressively decreased in control SCs after postnatal day 14, whereas levels were aberrantly elevated in Fbxw7-deleted SCs. Interestingly, the Gata-4 messenger RNA levels remained stable following Fbxw7 deletion. Fbxw7 silencing in SCs also induced progressive Leydig cell inefficiency and testosterone insufficiency. Collectively, these results demonstrate that Fbxw7 expression is required for SC maturation and function, potentially through degradation of GATA-4, to support pubertal testis development and spermatogenesis.

Chondrocyte mTORC1 activation stimulates miR-483-5p via HDAC4 in osteoarthritis progression.

The hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) in chondrocytes has been shown to accelerate the severity of destabilization of the medial meniscus-induced and age-related osteoarthritis (OA) phenotypes with aberrant chondrocyte hypertrophy and angiogenesis. Meanwhile, we previously reported that miR-483-5p is essential for the initiation and development of OA by stimulating chondrocyte hypertrophy and angiogenesis. The connection between mTORC1 and miR-483-5p activation in OA progression, however, remains unclear. In this study, we elucidated their relationship and identified the underlying mechanisms. The expression of miR-483-5p in the articular cartilage of cartilage-specific TSC1 knockout mice was assessed compared with control mice using the Agilent Mouse miRNA (8*60K) V19.0 array and real-time polymerase chain reaction (RT-PCR). The functional effects of the stimulation of miR-483-5p via histone deacetylase 4 (HDAC4) by mTORC1 in OA development, subsequently modulating its downstream targets matrilin 3 and tissue inhibitor of metalloproteinase 2, were examined by immunostaining, western blotting, and real-time PCR. This study revealed that miR-483-5p was responsible for mTORC1 activation-stimulated OA. Mechanistically, mTORC1 controls the HDAC4-dependent expression of miR-483-5p to stimulate chondrocyte hypertrophy, extracellular matrix degradation, and subchondral bone angiogenesis, and it consequently initiates and accelerates the development of OA. Our findings revealed a novel mTORC1-HDAC4-miR-483-5p pathway that is critical for OA development.