The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition.

BACKGROUND: Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. METHODS: To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. RESULTS: In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. CONCLUSIONS: Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

Foxp1 and Foxp4 Deletion Causes the Loss of Follicle Stem Cell Niche and Cyclic Hair Shedding by Inducing Inner Bulge Cell Apoptosis.

Quiescent hair follicle stem cells (HFSCs) reside in specialized bulge niche where they undergo activation and differentiation upon sensing niche-dependent signals during hair follicle (HF) homeostasis and wound repair. The underlying mechanism of HFSCs and bulge niche maintenance is poorly understood. Our previous study has reported that a transcription factor, forkhead box P1 (Foxp1), functions to maintain the quiescence of HFSCs. Here, we further discovered that forkhead box P4 (Foxp4), a close family member of Foxp1, had similar expression profiles in various components of HFs and formed a complex with Foxp1 in vitro and in vivo. The HF-specific deficiency of Foxp4 resulted in the precocious activation of HFSCs during hair cycles. In contrast to single Foxp1 or Foxp4 conditional knockout (cKO) mice, Foxp1/4 double cKO exerted an additive effect in the spectrum and severity of phenotypes in HFSC activation, hair cycling acceleration and hair loss, coupled with remarkable downregulation of fibroblast growth factor 18 (Fgf18) and bone morphogenetic protein 6 (Bmp6) expression in bulge cells. In addition, the double KO of Foxp1/4 induced the apoptosis of K6-positive (K6+) inner bulge cells, a well-established stem cell (SC) niche, thus resulting in the destruction of the bulge SC niche and recurrent hair loss. Our investigation reveals the synergistic role of Foxp1/4 in sustaining K6+ niche cells for the quiescence of HFSCs.

MiR-664-3p suppresses osteoblast differentiation and impairs bone formation via targeting Smad4 and Osterix.

Osteoporosis is a metabolic disorder characterized by low bone mass and deteriorated microarchitecture, with an increased risk of fracture. Some miRNAs have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass. Our miRNA sequencing results showed that miR-664-3p was significantly down-regulated during the osteogenic differentiation of the preosteoblast MC3T3-E1 cells. However, whether miR-664-3p has an impact on bone homeostasis remains unknown. In this study, we identified overexpression of miR-664-3p inhibited the osteoblast activity and matrix mineralization in vitro. Osteoblastic miR-664-3p transgenic mice exhibited reduced bone mass due to suppressed osteoblast function. Target prediction analysis and experimental validation confirmed Smad4 and Osterix (Osx) are the direct targets of miR-664-3p. Furthermore, specific inhibition of miR-664-3p by subperiosteal injection with miR-664-3p antagomir protected against ovariectomy-induced bone loss. In addition, miR-664-3p expression was markedly higher in the serum from patients with osteoporosis compared to that from normal subjects. Taken together, this study revealed that miR-664-3p suppressed osteogenesis and bone formation via targeting Smad4 and Osx. It also highlights the potential of miR-664-3p as a novel diagnostic and therapeutic target for osteoporotic patients.

MicroRNA-145 suppresses osteogenic differentiation of human jaw bone marrow mesenchymal stem cells partially via targeting semaphorin 3A.

Purpose: Human jaw bone marrow mesenchymal stem cells (h-JBMMSCs) are multipotent progenitor cells with osteogenic differentiation potential. MicroRNAs (miRNAs) have emerged as crucial modulators of osteoblast differentiation. In this study, we focus on the role of miR-145 and its target protein in osteoblast differentiation of h-JBMMSCs. Materials and Methods: h-JBMMSCs were isolated and cultured in osteogenic medium. miR-145 mimics and inhibitors were used to elevate and inhibit miR-145 expression, respectively. Osteogenic differentiation was determined by Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining, and osteogenic marker detection using quantitative real-time reverse transcription PCR (qRT-PCR) assay. Bioinformatic analysis and luciferase reporter assay were used to identify the target gene of miR-145. Results: MiR-145 was down-regulated during osteogenesis of h-JBMMSCs. Inhibition of miR-145 promoted osteogenic differentiation of h-JBMMSCs, revealed by enhanced activity of alkaline phosphatase (ALP), greater mineralisation, and increased expression levels of the osteogenic markers, such as Runt-related transcription factor 2 (RUNX2), Osterix (OSX), ALP and COL1A1. MiR-145 could negatively regulate semaphorin3A (SEMA3A), which acts as a positive regulator of osteogenesis. MiR-145 inhibitor induced osteogenesis could be partially attenuated by SEMA3A siRNA treatment in h-JBMMSCs. Conclusions: Our data show that miR-145 directly targets SEMA3A, and also suggest miR-145 as a suppressor, plays an important role in the osteogenic differentiation of h-JBMMSCs.

αB-crystallin (CRYAB) regulates the proliferation, apoptosis, synthesis and degradation of extracellular matrix of chondrocytes in osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease and hard to cure at present. Alpha B-crystallin (CRYAB) has been identified as a downregulated gene in OA cartilage. However, the precise roles and underlying molecular mechanisms of CRYAB in OA progression have not been elucidated. In the present study, we found that the expression of CRYAB in cartilages from patients with OA was significantly lower than that in the cartilages from patients with no prior medical history of OA. We established mouse models with OA by destabilization of the medial meniscus (DMM) surgery and found that the expression of CRYAB in OA cartilage was lower than that in the normal cartilages, too. Moreover, we demonstrated that the expression of CRYAB was increased during chondrogenic differentiation and cartilage development. Functional assays revealed that overexpression of CRYAB promoted the proliferation of chondrocytes and inhibited apoptosis, while knockdown of CRYAB presented opposite results. In addition, overexpression of CRYAB upregulated the expression of anabolic markers, Col2a1 and ACAN, and reduced the expression of catabolic markers, MMP13 and ADAMTS5. Conversely, knockdown of CRYAB blocked the expression of the anabolic markers and increased the expression of catabolic markers. Collectively, the results suggest that CRYAB promoted the proliferation and extracellular matrix production of chondrocytes, and inhibited chondrocytes apoptosis and cartilage degradation simultaneously. Thus, CRYAB might be a potential therapeutic target for OA treatment.

Establishment and characterization of an immortalized human chondrocyte cell line.

OBJECTIVES: Following a specific number of mitotic divisions, primary chondrocytes undergo proliferative senescence, thwarting efforts to expand sufficient populations in vitro suitable to meet the needs of scientific research or medical therapies. Therefore, the human telomerase reverse transcriptase (TERT) was used to immortalize human chondrocyte and establish a cell line that escape from cellular senescence. RESULTS: The human chondrocytes were successfully immortalized by ectopic stable expression of TERT. The established TERT-Chondrocyte cell line showed robust proliferation capacity, even in late passages up to P20, and displayed little cellular senescence. Moreover, TERT-Chondrocyte cells at 20th passage showed similar chondrocyte properties to normal chondrocytes at early passages. CONCLUSIONS: Ectopic stable expression of TERT is an effective way to immortalized human chondrocyte. The immortalized chondrocytes displayed little cellular senescence, showed promise as an in vitro model to investigate osteoarthritis, and may be a promising resource for cell-based therapy for damaged cartilage.

Osterix promotes the migration and angiogenesis of breast cancer by upregulation of S100A4 expression.

As a key transcription factor required for bone formation, osterix (OSX) has been reported to be overexpressed in various cancers, however, its roles in breast cancer progression remain poorly understood. In this study, we demonstrated that OSX was highly expressed in metastatic breast cancer cells. Moreover, it could upregulate the expression of S100 calcium binding protein A4 (S100A4) and potentiate breast cancer cell migration and tumor angiogenesis in vitro and in vivo. Importantly, inhibition of S100A4 impaired OSX-induced cell migration and capillary-like tube formation. Restored S100A4 expression rescued OSX-short hairpin RNA-suppressed cell migration and capillary-like tube formation. Moreover, the expression levels of OSX and S100A4 correlated significantly in human breast tumors. Our study suggested that OSX acts as an oncogenic driver in cell migration and tumor angiogenesis, and may serve as a potential therapeutic target for human breast cancer treatment.

Osterix Decreases the Chemosensitivity of Breast Cancer Cells by Upregulating GALNT14.

BACKGROUND/AIMS: Osterix (Osx), a key regulator of osteoblast differentiation and bone formation, has been recently reported to be associated with the progression of breast cancer. However, the precise roles of Osx in breast cancer remain unclear. METHODS: Drug sensitivity of the cancer cells was assessed using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Target genes were obtained by high-throughput Illumina sequencing and were confirmed in vitro and in vivo. Apoptosis was analysed by Hoechst staining and western blotting. A tissue microarray including 129 samples from breast cancer patients was used for immunohistochemistry (IHC) assays. RESULTS: Overexpression of Osx decreased the chemosensitivity of breast cancer cells, while knockdown of Osx increased the chemosensitivity of breast cancer cells. In particular, we found that the decreased chemosensitivity effect was significantly associated with elevated expression of the polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14). Silencing of GALNT14 in Osx-overexpressed cells restored the decreased chemosensitivity. Conversely, overexpression of GALNT14 in Osx-knockdown cells abrogated the increased chemosensitivity in breast cancer cells. In addition, we revealed that Osx decreased GALNT14-dependent chemosensitivity by enhancing anti-apoptosis. GALNT14 expression exhibited a significant association with breast cancer stages as well as the disease-free survival (DFS) rate. CONCLUSION: Osx plays an important role in the chemosensitivity and inhibition of Osx expression may represent a therapeutic strategy to enhance the chemosensitivity of breast cancer.

The upregulation of circFoxp1 influences keloid by promoting cell proliferation.

As a result of abnormal wound healing in susceptible individuals, keloids are characterized by hyperproliferation of fibroblasts and excessive deposition of the extracellular matrix (ECM). Current surgical and therapeutic modalities provide limited satisfactory results. Circular ribonucleic acids (circRNAs) play a crucial role in the pathogenesis of various fibrotic diseases, but the potential biological function and expression profile of circRNAs in keloid formation remain unknown. In this study, we explored the function of circFoxp1 on keloid formation. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results revealed that circFoxp1 expression was higher in the keloid tissues. Furthermore, RNA-fluorescence in situ hybridization (RNA-FISH) and RNAscope illustrated that circFoxp1 was present in the cytoplasm. Subsequent cellular experiments demonstrated that circFoxp1 overexpression enhanced proliferation, migration, and ECM deposition. In addition, apoptosis was inhibited. Cell proliferation, inflammatory response, and oxidative phosphorylation of fibroblasts were also observed by RNA sequencing and were closely related to scar formation. The therapeutic potential of circFoxp1 was investigated by establishing keloid implantation models. In vivo, circFoxp1 can promote fibroblast proliferation and ECM deposition. RNA pull-down and western blot assays verified the interaction of circFoxp1 with RACK1. The present study reveals that circFoxp1 contributes to the pathological hyperplasia of keloid, which may improve inflammation and cell proliferation. Our data indicate that circFoxp1 may serve as a novel, promising therapeutic target, presenting a new avenue for understanding the underlying pathogenesis of keloid.

Upregulated osterix promotes invasion and bone metastasis and predicts for a poor prognosis in breast cancer.

Approximately 70% of patients with advanced breast cancer develop bone metastases, accompanied by complications, such as bone pain, fracture, and hypercalcemia. However, our understanding of the molecular mechanisms that govern this process remains fragmentary. Osterix (Osx) is a zinc finger-containing transcription factor essential for osteoblast differentiation and bone formation. Here, we identified the functional roles of Osx in facilitating breast cancer invasion and bone metastasis. Osx upregulation was associated with lymph node metastasis and was negatively prognostic for overall survival. Knockdown of Osx inhibited invasion of breast cancer and osteolytic metastasis by downregulating MMP9, MMP13, VEGF, IL-8, and PTHrP, which are involved in invasion, angiogenesis, and osteolysis; overexpression of Osx had the opposite effect. Moreover, MMP9 was a direct target of Osx and mediated the Osx-driven invasion of breast cancer cells. Together, our data showed that Osx facilitates bone metastasis of breast cancer by upregulating the expression of a cohort of genes that contribute to steps in the metastatic cascade. These findings suggest that Osx is an attractive target for the control of bone metastasis of breast cancers.

miR-130b-3p inhibits cell invasion and migration by targeting the Notch ligand Delta-like 1 in breast carcinoma.

Breast carcinoma is the most common malignancy in women, and the incidence rate has increased dramatically in recent years. Metastasis is responsible for most advanced breast cancer mortality, but the underlying mechanisms remain poorly understood despite extensive research. Recently, short non-coding RNA molecules, including miRNAs, which mediate changes in signalling pathways, have emerged as metastatic regulators of the breast carcinoma. Previous reports have suggested that miR-130b-3p has both oncogenic and tumour suppressor functions in a cancer type-dependent manner. However, the roles and underlying molecular mechanisms of miR-130b-3p in the development of metastasis in breast carcinoma remain unclear. Here, we reported for the first time that miR-130b-3p was differentially expressed in early-stage non-invasive MCF-7 human breast carcinoma cells and aggressive late-stage MDA-MB-231 cells. In gain-of-function and loss-of-function studies, we demonstrated that miR-130b-3p could inhibit breast carcinoma cell invasion and migration by directly targeting the Notch ligand Delta-like 1 (DLL1). Our data also indicated that MMP-9, MMP-13, and VEGF were regulated by miR-130b-3p and may be involved in the inhibition of cell invasion and migration in breast carcinoma. Collectively, our findings reveal a new regulatory mechanism of miR-130b-3p and suggest that miR-130b-3p may be a potential target against human breast cancer metastasis.