Lgr4 promotes aerobic glycolysis and differentiation in osteoblasts via the canonical Wnt/β-catenin pathway

Lgr4, a G-protein-coupled receptor, is associated with various physiological and pathological processes including oncogenesis, energy metabolism, and bone remodeling. However, whether Lgr4 is involved in osteoblasts' metabolism is not clear. Here we uncover that in preosteoblast cell line, lacking Lgr4 results in decreased osteogenic function along with reduced glucose consumption, glucose uptake, and lactate production in the presence of abundant oxygen, which is referred to as aerobic glycolysis. Activating canonical Wnt/β-catenin signaling rescued the glycolytic dysfunction. Lgr4 promotes the expression of pyruvate dehydrogenase kinase 1 (pdk1) and is abolished by interfering canonical Wnt/β-catenin signaling. Mice lacking Lgr4 specifically in osteoblasts (Lgr4^osb−/−) exhibit decreased bone mass and strength due to reduced bone formation. Additionally, glycolysis of osteoblasts is impaired in Lgr4^osb−/− mice. Our study reveals a novel function of Lgr4 in regulating the cellular metabolism of osteoblasts. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Irradiation by high-intensity red light-emitting diode enhances human bone marrow mesenchymal stem cells osteogenic differentiation and mineralization through Wnt/β-catenin signaling pathway

Lasers in Medical Science - Photobiomodulation therapy (PBMT) using a light-emitting diode (LED) has been employed for various photomedicine studies. The aim of this study was to determine the...     

Comparison of gene expression of the oncogenic Wnt/β-catenin signaling pathway components in the mouse and human epididymis

β-catenin is an integral part of the Wnt signaling pathway and has been linked to tumorigenesis and multiple developmental processes. The high β-catenin expression with low tumor incidence in the human epididymis is thus intriguing. In the present study, the β-catenin gene and protein was found to be highly expressed in the murine caput epididymidis, and the protein mainly localized along the lateral plasma membranes of adjacent epithelial cells throughout both human and mouse epididymides. Furthermore, the adult mouse epididymis was found to express almost all the Wnt/β-catenin signaling pathway genes that were determined previously by our group in the human organ. Despite the differences in epididymal structure, the similar location of β-catenin and the high concordance of this pathway''s components’ gene expression in both the adult human and mouse epididymides make the mouse a suitable animal model for studying the anti-tumor mechanism of the epididymis. In addition, both the mRNA and protein expression of β-catenin shared a similar spatial expression as the mRNA of Ros1, a proto-oncogene and a key developmental regulator of the initial segment of the mouse epididymis. The observations on the parallel temporal expression of β-catenin and Ros1 during postnatal development raise the possibility that the canonical Wnt signaling pathway has an additional role in the postnatal development of mouse epididymis.     

Roles of Wnt/β-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats

Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt–β-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt–β-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed β-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200 mg/kg/day for 8 days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n = 8, P < 0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P < 0.01). At 25 mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/β-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury.     

Serum extracellular secreted antagonists of the canonical Wnt/β-catenin signaling pathway in patients with Cushing’s syndrome

Summary

Patients with endogenous hypercortisolism have higher sclerostin, but do not differ in Dickkopf 1 (Dkk1) or secreted frizzled-related protein 1 (SFRP1) levels as compared to healthy control.

Introduction

Endogenous Cushing’s syndrome (CS), usually affecting young and otherwise healthy patients, is a good model to validate the effects of supraphysiological levels of glucocorticoids in humans. This study evaluates circulating levels of extracellular antagonists of the Wnt/β-catenin signaling pathway (sclerostin, Dkk1, SFRP1) in patients with CS versus healthy individuals.

Methods

Forty patients with clinically and biochemically evident CS and 40 sex-, age-, and body mass index-matched healthy subjects provided fasting serum samples for sclerostin, SFRP1 and Dkk1, along with bone turnover markers.

Results

Patients with CS had higher sclerostin levels (34.5 (30.3–37.1) pmol/L) versus healthy individuals (29.9 (24.3–36.8) pmol/L) (p?=?0.032). Differences in sclerostin were due to the lack of lower sclerostin values rather than an increase in protein levels above the upper limits of the healthy control. The odds of sclerostin levels being higher than 30 pmol/L were greater in patients with CS as compared with the odds in healthy subjects (odds ratio?=?3.81 95 % confidence interval 1.45–10.02) (p?=?0.01). It coexisted with suppressed bone formation and unchanged bone resorption markers. Dkk1, SFRP1 did not differ from the control group.

Conclusions

Of all the tested proteins (sclerostin, Dkk1, SFRP1), only sclerostin showed a significant difference when contrasting CS with healthy subjects. Hypercortisolism might prevent the down-regulation of sclerostin. Targeting sclerostin seems to be a promising therapeutic approach to treating osteoporosis in patients with CS.     

Effect of Wnt/β-catenin signals on the fracture healing in transgenic mice

Objective To evaluate the effect of Wnt/β-catenin signals on the fracture healing in transgenic mice. Methods Col2al-ICAT transgenic mice were generated by the gene targeting technology with the ICAT transgene specifically expressed in chondrocytes as a competitive inhibitor to block Wnt/ β-catenin signals. The 8-week-old Col2al-ICAT mice were used in the experimental group and the wild type (WT) littermates with the same age served as the control group. A transverse osteotomy was performed at the middle of the tibia and the fracture healing was evaluated on the 7th, 9th, 14th, 21th and 28th days re-spectively after fracture. Roentgenogyaphy and histology observations were performed to evaluate the fracture healing pattern and the histomorphometric analysis was used to quantitate the cartilage callus volume / total callus volume (CV/TV) or bony callus volume / total callus volume (BV/TV). Results X-ray exam-ination revealed that on the 21st day after fracture, callus appeared at the fracture gap to form a bony bridge in WT mice while a radiolucent zone was apparent in the fracture gap in the Col2al-ICAT transgenic mice. Histology observation revealed that compared with WT mice, the formation of cartilage callus and endochondral ossification were delayed in Col2al-ICAT transgenic mice. Histomorphometric analysis indicated that the peak value of CV/TV arrived later in Col2al-ICAT transgenie mice than in WT mice. The BV/TV in Col2al-ICAT transgenic mice was significantly less than that in WT mice on 14th and 21st days after fracture (P<0.05). Conclusion The Wnt/β-catenin signals cause delayed fracture healing in Col2al-ICAT transgenic mice by affecting the cartilage callus formation and endochondral ossification.     

Imbalance in the estrogen/androgen ratio may affect prostate fibrosis through the TGF-β/Smad signaling pathway

Objective

The present study aimed to investigate the effects of an imbalance in the estrogen/androgen ratio on prostate fibrosis.

Methods

Different concentrations of dihydrotestosterone (DHT) or estradiol (E₂) dissolved in corn oil were injected subcutaneously into the nape of the castrated Sprague–Dawley (SD) rats over 28 consecutive days. Masson’s trichrome staining and immunohistochemical staining were performed to detect the content of collagen fibers and the expression of collagen I, fibronectin, and elastin in the rat prostate of each group, respectively. DHT?+?E₂ at different concentrations was administered to human normal prostate stromal immortalized cells (WPMY-1 cells) for 1 week. The expression of collagen I, fibronectin, elastin, transforming growth factor-β₁ (TGF-β₁), Smad3, and Smad7 was detected by Western blotting (WB). Then, WPMY-1 cells treated with 10 nM DHT?+?5 pM E₂ were incubated with the TGF-β/Smad pathway inhibitor SD208 for 1 week, after which collagen I, fibronectin, and elastin expression was detected by WB.

Results

Compared with the uncastrated control and corn oil injection groups, the collagen fiber content and collagen I and fibronectin expression were increased and elastin expression was decreased in the castrated rat prostate with corn oil injection group (p?<?0.01). Compared to castrated corn oil injection group, collagen fiber content, collagen I, and fibronectin expression were significantly decreased, and elastin expression was significantly increased in the castrated rat prostate 0.15 mg/kg DHT treatment group (p?<?0.01). Following treatment with 0.15 mg/kg DHT, the content of collagen fibers, and the expression of collagen I and fibronectin were increased, and the expression of elastin was decreased in the rat prostate with increasing concentrations of E₂ treatment group compared to the 0.15 mg/kg DHT group (p?<?0.05, p?<?0.01). Following treatment with 0.05 mg/kg E₂, the collagen fiber content and the expression of collagen I and fibronectin were decreased, and the expression of elastin was increased in the rat prostate with increasing DHT concentration treatment group compared to the 0.05 mg/kg E₂ group (p?<?0.05, p?<?0.01). Compared with the Control group, the expression of collagen I, fibronectin, TGF-β₁ and Smad3 was decreased, and the expression of elastin and Smad7 was increased in WPMY-1 cells after treatment with 10 nM DHT (p?<?0.01). Following treatment with 10 nM DHT, the expression of collagen I, fibronectin, TGF-β₁, and Smad3 was increased, and the expression of elastin and Smad7 was decreased in WPMY-1 cells with increasing E₂ concentration treatment compared to the 10 nM DHT group (p?<?0.05, p?<?0.01). Following treatment with 5 pM E₂, the expression of collagen I, fibronectin, TGF-β₁, and Smad3 was decreased, and elastin and Smad7 expression was increased with increasing DHT concentration compared to the 5 pM E₂ group (p?<?0.05, p?<?0.01). Compared to the 10 nM DHT?+?5 pM E₂ group, the expressions of collagen I and fibronectin were decreased; the expression of elastin was increased in WPMY-1 cells after the supplement of TGF-β/Smad pathway inhibitor SD208 group (p?<?0.05, p?<?0.01).

Conclusions

An imbalance in the estrogen/androgen ratio may affect prostate fibrosis. E₂ may activate the degree of prostate fibrosis. In contrast to the effect of E₂, DHT may inhibit the degree of prostate fibrosis, which might involve the TGF-β/Smad signaling pathway.

     

Attenuation of the degenerative effects of endothelin-1 on cartilaginous end plate cells by the endothelin receptor antagonist BQ-123 via the Wnt/β-catenin signaling pathway

Background Context

Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown.

Repression of osteocyte Wnt/β-catenin signaling is an early event in the progression of renal osteodystrophy

Chronic kidney disease-mineral bone disorder (CKD-MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft-tissue calcification. Emerging evidence suggests that features of CKD-MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild-type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild-type mice. To capture the early molecular and cellular events in the progression of CKD-MBD we examined cell-specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor-Ser33/37-β-catenin was observed both in mouse and human bones. Overall repression of Wnt/β-catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF-κB ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late-stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD-MBD and suggest that repression of the Wnt/β-catenin pathway is involved in the pathogenesis of renal osteodystrophy.     

lncRNA FER1L4调控Wnt/β-catenin信号通路对甲状腺癌细胞恶性生物学行为的影响

目的：探讨lncRNA FER1L4调控Wnt/β-cate nin信号通路对甲状腺癌细胞恶性生物学行为的影响。方法：检测人甲状腺滤泡上皮正常细胞和甲状腺癌细胞中FER1L4的表达水平。将SW579细胞分为control组、s i-NC组、s i-FER1L4组、Li Cl+s i-FER1L4组;检测各转染组细胞FER1L4的表达水平,细胞增殖,细胞凋亡率,细胞迁移和侵袭;检测细胞Cleaved caspase3、β-catenin、c-Myc和Cyclin D1蛋白表达水平。结果：SW579细胞中FER1L4表达水平最显著;干扰FER1L4后,其细胞活力、增殖率、迁移和侵袭个数、β-catenin、c-Myc和Cyclin D1蛋白表达显著降低,细胞凋亡率和Cleaved caspase3表达显著升高（P<0.05）;与si-FER1L4组相比,Li Cl+si-FER1L4组细胞活力、增殖率、迁移和侵袭个数、β-catenin、c-Myc和Cyclin D1蛋白表达显著升高,细胞凋亡率和Cleaved cas pas e3表达显著降低（P<0.05）。结论：干扰FER...     

β-catenin (CTNNB1) S33C mutation in ovarian microcystic stromal tumors

Microcystic stromal tumor (MCST) is a recently described subtype of ovarian tumor characterized by prominent microcystic histologic pattern and diffuse immunoreactivity for CD10 and vimentin. However, its pathobiology, particularly its histogenesis, remains largely unclear. Here, we report 2 cases of ovarian MCST, in which we have performed extensive histologic, immunohistochemical, and genetic investigations to determine its distinct nature among ovarian neoplasms. The patients were 32 and 41 years of age. Both tumors were solid and cystic masses involving the right ovary. Microscopically, tumor cells with generally bland, round-to-ovoid nuclei grew in microcystic, macrocystic, and solid patterns. Intervening thick fibrous stroma was observed. Immunohistochemically, tumor cells were diffusely and strongly positive for CD10, vimentin, and Wilms tumor 1. Furthermore, we detected aberrant nuclear expression of β-catenin protein in both cases. Of interest, mutation analyses revealed the presence of an identical point mutation, c.98C>G, in exon 3 of β-catenin (CTNNB1) in both tumors. This is an oncogenic mutation that causes replacement of serine with cysteine at codon 33, leading to the loss of a phosphorylation site in the β-catenin protein. The results of this study strongly suggest that dysregulation of the Wnt/β-catenin pathway plays a fundamental role in the pathogenesis of ovarian MCST. Finally, by comparing the immunophenotype of MCST with its histologic mimics and other ovarian sex cord-stromal tumors, we were able to identify unique features of MCST and a panel of markers useful in differential diagnosis.     

Smad7 inhibits TGF-β1-induced MCP-1 upregulation through a MAPK/p38 pathway in rat peritoneal mesothelial cells

Purpose

The TGF-β1/Smad signaling pathway is subject to inhibition by Smad7. High expression of Smad7 in the peritoneum of rats can delay and attenuate not only peritoneal fibrosis, but also monocyte infiltration into the peritoneum. The aim of this study was to investigate the anti-inflammatory mechanism of Smad7 in peritoneal fibrosis.

Methods

Rat peritoneal mesothelial cells were stimulated with TGF-β1, and the expression of MCP-1 protein and mRNA was measured. Furthermore, the expression of MCP-1 was determined following inhibition of TGF-β/Smad or p38 signaling using Smad7 transfection or SB203580 (10 μmol/L), respectively. The effect of exogenous Smad7 and SB203580 on activation of the TGF-β/Smad and p38 signaling pathways was also studied.

Results

TGF-β1 significantly upregulated the expression of MCP-1 at both the protein and mRNA level in a time-dependent manner. Exogenous Smad7 and SB203580 markedly inhibited TGF-β1-induced MCP-1 expression. Moreover, high expression of exogenous Smad7 not only inhibited phosphorylation of Smad2 and Smad3, but also diminished the level of phosphorylated p38. However, SB203580 had no effect on the phosphorylation of Smad2 and Smad3.

Conclusions

TGF-β1 exhibits pro-inflammatory effects through the upregulation of MCP-1 in peritoneal fibrosis. Smad7 inhibits TGF-β1 induced MCP-1 upregulation through a MAPK/p38-dependent pathway.     

Low sirtuin 1 levels in human osteoarthritis subchondral osteoblasts lead to abnormal sclerostin expression which decreases Wnt/β-catenin activity

IntroductionWnt/β-catenin (cWnt) signaling plays a key role in osteogenesis by promoting the differentiation and mineralization of osteoblasts, activities altered in human osteoarthritic subchondral osteoblast (OA Ob). Sclerostin (SOST) has been shown to alter cWnt signaling. Sirtuin 1 (SIRT1) acts as a novel bone regulator and represses SOST levels in Ob. However the role of SIRT1 and SOST in OA Ob remains unknown. Herein, we explored the role played by SIRT1 and SOST on the abnormal mineralization and cWnt signaling in OA Ob.MethodsPrimary human normal and OA Ob were prepared from tibial plateaus. SOST levels were evaluated by immunohistochemistry, the expression and production of genes by qRT-PCR and WB analysis. Their inhibitions were performed using siRNA. cWnt signaling was measured by the TOPflash TCF/lef luciferase reporter assay. Mineralization was determined by alizarin red staining.ResultsSOST levels were significantly increased in OA Ob compared to normal and were linked with elevated TGF-β1 levels in these cells. SIRT1 expression was significantly reduced in OA Ob compared to normal yet not modified by TGF-β1. Specific inhibition of SIRT1 increased TGF-β1 and SOST expressions in OA Ob, while stimulating SIRT1 activity with β-Nicotinamide mononucleotide reduced the expression of TGF-β1 and SOST, and increased mineralization in OA Ob. Resveratrol also reduced SOST expression in OA Ob. Reduced cWnt signaling, β-catenin levels, and mineralization in OA Ob were all corrected via reducing SOST expression.ConclusionThese data indicate that high level of SOST is responsible, in part, for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.