TGF-ß3 mediates mitochondrial dynamics through the p-Smad3/AMPK pathway.

It is well recognized that mitochondrial dynamics plays a vital role in cartilage physiology. Any perturbation in mitochondrial dynamics could cause disorders in cartilage metabolism and even lead to the occurrence of cartilage diseases such as osteoarthritis (OA). TGF-ß3, as an important growth factor that appears in the joints of OA disease, shows its great potential in chondrocyte growth and metabolism. Nevertheless, the role of TGF-ß3 on mitochondrial dynamics is still not well understood. Here we aimed to investigate the effect of TGF-ß3 on mitochondrial dynamics of chondrocytes and reveal its underlying bio-mechanism. By using transmission electron microscopy (TEM) for the number and morphology of mitochondria, western blotting for the protein expressions, immunofluorescence for the cytoplasmic distributions of proteins, and RNA sequencing for the transcriptome changes related to mitochondrial dynamics. We found that TGF-ß3 could increase the number of mitochondria in chondrocytes. TGF-ß3-enhanced mitochondrial number was via promoting the mitochondrial fission. The mitochondrial fission induced by TGF-ß3 was mediated by AMPK signaling. TGF-ß3 activated canonical p-Smad3 signaling and resultantly mediated AMPK-induced mitochondrial fission. Taken together, these results elucidate an understanding of the role of TGF-ß3 on mitochondrial dynamics in chondrocytes and provide potential cues for therapeutic strategies in cartilage injury and OA disease in terms of energy metabolism.

FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway.

Fibroblast growth factor 19 (FGF19) is recognized to play an essential role in cartilage development and physiology, and has emerged as a potential therapeutic target for skeletal metabolic diseases. However, FGF19-mediated cellular behavior in chondrocytes remains a big challenge. In the current study, we aimed to investigate the role of FGF19 on chondrocytes by characterizing mitochondrial biogenesis and fission-fusion dynamic equilibrium and exploring the underlying mechanism. We first found that FGF19 enhanced mitochondrial biogenesis in chondrocytes with the help of ß Klotho (KLB), a vital accessory protein for assisting the binding of FGF19 to its receptor, and the enhanced biogenesis accompanied with a fusion of mitochondria, reflecting in the elongation of individual mitochondria and the up-regulation of mitochondrial fusion proteins. We then revealed that FGF19-mediated mitochondrial biogenesis and fusion required the binding of FGF19 to the membrane receptor, FGFR4, and the activation of AMP-activated protein kinase alpha (AMPKα)/peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α)/sirtuin 1 (SIRT1) axis. Finally, we demonstrated that FGF19-mediated mitochondrial biogenesis and fusion was mainly dependent on the activation of p-p38 signaling. Inhibition of p38 signaling largely reduced the high expression of AMPKα/PGC-1α/SIRT1 axis, decreased the up-regulation of mitochondrial fusion proteins and impaired the enhancement of mitochondrial network morphology in chondrocytes induced by FGF19. Taking together, our results indicate that FGF19 could increase mitochondrial biogenesis and fusion via AMPKα-p38/MAPK signaling, which enlarge the understanding of FGF19 on chondrocyte metabolism. Video Abstract.

IL-10 enhances cell-to-cell communication in chondrocytes via STAT3 signaling pathway.

Gap junction intercellular communication (GJIC) allows the transfer of material, message and energy between cells, which influences cell behaviors including cell proliferation, migration, differentiation and apoptosis and determines cell fate. Interleukin-10 (IL-10), a versatile cytokine, attracts more and more attention in the cartilage pathology such as osteoarthritis (OA) due to its potential in anti-inflammation and wound repair. However, whether IL-10 can mediate GJIC in chondrocytes remains elusive. In the current study, we aimed to explore the role of IL-10 on GJIC and its underlying mechanism. We found that IL-10 can promote GJIC in living chondrocytes. IL-10-enhanced GJIC in chondrocytes was dependent on the up-regulation of connexin 43 (Cx43). Knockdown experiment based on siRNA interference then confirmed that IL-10-enhanced GJIC required participation of IL-10 receptor 1 (IL-10R1). IL-10 activated signal transducer and activator of transcription 3 (STAT3) signaling and promoted the nuclear accumulation of p-STAT3 through IL-10 receptor 1. Inhibitor experiment further confirmed the importance of STAT3 signaling in IL-10-mediated GJIC. Taking together, our results provided a thorough process of IL-10-modulated cell-to-cell communication in chondrocytes and established a bridge between inflammatory factor, IL-10, and GJIC, which can increase our understanding about the physiology and pathology of cartilage.

TGF-ß2 increases cell-cell communication in chondrocytes via p-Smad3 signalling.

Connexin 43 (Cx43)-mediated gap junction intercellular communication (GJIC) plays a crucial role in the pathology and physiology of joint tissues. Transforming growth factor-ß2 (TGF-ß2), one of the potent regulatory factors in chondrocytes, plays a key role in the regulation of cell cycle and development of joint diseases. However, it is still unknown how TGF-ß2 mediates GJIC in chondrocytes. The aim of this study was to explore the potential mechanism by which TGF-ß2 regulates GJIC in chondrocytes. CCK-8 assays and scratch assays were performed to define the role of TGF-ß2 on cell proliferation and migration. The scrape loading/dye transfer assay and scanning electron microscopy (SEM) were used to verify the effect of TGF-ß2 on GJIC between chondrocytes. qPCR was performed to analyse the expression of genes in the gap junction protein family in chondrocytes. The expression of the Cx43 protein and phosphorylated Smad3 (p-Smad3) was evaluated by western blot assay. Immunofluorescence staining was used to explore p-Smad3 signalling pathway activation and Cx43 distribution. From these experiments, we found that the Cx43 protein was the most highly expressed member of the gap junction protein family in chondrocytes. We also found that TGF-ß2 facilitated cell-to-cell communication in chondrocytes by upregulating Cx43 expression in chondrocytes. Finally, we found that TGF-ß2 activated Smad3 signalling and promoted the nuclear aggregation of p-Smad3. Inhibition experiments by SIS3 also confirmed that TGF-ß2-mediated GJIC through p-Smad3 signalling. For the first time, this study confirmed that TGF-ß2 could regulate the formation of Cx43-mediated GJIC in chondrocytes via the canonical p-Smad3 signalling pathway.

Role of Mitochondria in Physiology of Chondrocytes and Diseases of Osteoarthritis and Rheumatoid Arthritis.

PURPOSE OF REVIEW: Mitochondria are recognized to be one of the most important organelles in chondrocytes for their role in triphosphate (ATP) generation through aerobic phosphorylation. Mitochondria also participate in many intracellular processes involving modulating reactive oxygen species (ROS), responding to instantaneous hypoxia stress, regulating cytoplasmic transport of calcium ion, and directing mitophagy to maintain the homeostasis of individual chondrocytes. DESIGNS: To summarize the specific role of mitochondria in chondrocytes, we screened related papers in PubMed database and the search strategy is ((mitochondria) AND (chondrocyte)) AND (English [Language]). The articles published in the past 5 years were included and 130 papers were studied. RESULTS: In recent years, the integrity of mitochondrial structure has been regarded as a prerequisite for normal chondrocyte survival and defect in mitochondrial function has been found in cartilage-related diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA). However, the understanding of mitochondria in cartilage is still largely limited. The mechanism on how the changes in mitochondrial structure and function directly lead to the occurrence and development of cartilage-related diseases remains to be elusive. CONCLUSION: This review aims to summarize the role of mitochondria in chondrocytes under the physiological and pathological changes from ATP generation, calcium homeostasis, redox regulation, mitophagy modulation, mitochondria biogenesis to immune response activation. The enhanced understanding of molecular mechanisms in mitochondria might offer some new cues for cartilage remodeling and pathological intervention.

PDGF-AA promotes cell-to-cell communication in osteocytes through PI3K/Akt signaling pathway.

Osteocytes are the main sensitive cells in bone remodeling due to their potent functional cell processes from the mineralized bone matrix to the bone surface and the bone marrow. Neighboring osteocytes communicate with each other by these cell processes to achieve molecular exchange through gap junction channels. Platelet-derived growth factor-AA (PDGF-AA) has been reported to enhance bone tissue remodeling by promoting cell proliferation, migration, and autocrine secretion in osteoid cell linage. However, the effect of PDGF-AA on intercellular communication between osteocytes is still unclear. In the present study, we elucidated that PDGF-AA could enhance the formation of dendritic processes of osteocytes and the gap junctional intercellular communication by promoting the expression of connexin43 (Cx43). This modulation process was mainly dependent on the activation of phosphorylation of Akt protein by phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling. Inhibition of PI3K/Akt signaling decreased the Cx43 expression induced by PDGF-AA. These results establish a bridge between PDGF-AA and cell-cell communication in osteocytes, which could help us understand the molecular exchange between bone cells and fracture healing.

Identification of novel susceptibility loci for non-syndromic cleft lip with or without cleft palate.

Although several genome-wide association studies (GWAS) of non-syndromic cleft lip with or without cleft palate (NSCL/P) have been reported, more novel association signals are remained to be exploited. Here, we performed an in-depth analysis of our previously published Chinese GWAS cohort study with replication in an extra dbGaP case-parent trios and another in-house Nanjing cohort, and finally identified five novel significant association signals (rs11119445: 3' of SERTAD4, P = 6.44 × 10-14 ; rs227227 and rs12561877: intron of SYT14, P = 5.02 × 10-13 and 2.80 × 10-11 , respectively; rs643118: intron of TRAF3IP3, P = 4.45 × 10-6 ; rs2095293: intron of NR6A1, P = 2.98 × 10-5 ). The mean (standard deviation) of the weighted genetic risk score (wGRS) from these SNPs was 1.83 (0.65) for NSCL/P cases and 1.58 (0.68) for controls, respectively (P = 2.67 × 10-16 ). Rs643118 was identified as a shared susceptible factor of NSCL/P among Asians and Europeans, while rs227227 may contribute to the risk of NSCL/P as well as NSCPO. In addition, sertad4 knockdown zebrafish models resulted in down-regulation of sox2 and caused oedema around the heart and mandibular deficiency, compared with control embryos. Taken together, this study has improved our understanding of the genetic susceptibility to NSCL/P and provided further clues to its aetiology in the Chinese population.

Association Study of Genetic Variants in Autophagy Pathway and Risk of Non-syndromic Cleft Lip With or Without Cleft Palate.

Although genetic variants in autophagy pathway genes were associated with the risk of oral cancers and early development in embryos, their associations with non-syndromic cleft lip with or without cleft palate (NSCL/P) risk remained unclear. A two-stage case-control study (2,027 NSCL/P cases and 1,843 controls) was performed to investigate the associations between single nucleotide polymorphisms (SNPs) in 23 autophagy pathway genes and NSCL/P susceptibility. The logistic regression model was used to calculate effects of SNPs on NSCL/P susceptibility. Gene-based analysis was performed via the sequence kernel association test (SKAT) and multi-marker analysis of genomic annotation (MAGMA) methods. Expression quantitative trait loci (eQTL) analysis was conducted using NSCL/P lip tissue samples. Gene expression during embryonic development was evaluated using RNA-Seq. Functional roles were explored by luciferase activity assay, cell apoptosis, proliferation, and cycle in vitro. Rs2301104 in HIF1A was significantly associated with NSCL/P susceptibility in the combined analysis (OR: 1.29, 95% CI: 1.09-1.29, P = 3.39 × 10-03), and showed strong evidence of association heterogeneity (P = 9.06 × 10-03) with obvious association in the female (OR: 1.80; 95% CI: 1.32-2.45; P = 1.79 × 10-04). The G allele of rs2301104 was associated with enhanced transcription activity and high expression of HIF1A compared with that of C allele. Moreover, rs2301104 exhibited an eQTL effect for HIF1A with its GC/CC genotypes associated with decreased HIF1A expression compared with those with GG genotypes (P = 3.1 × 10-2). Knockdown of HIF1A induced cell apoptosis and inhibited cell proliferation in human embryonic palate mesenchyme (HEPM) and human oral epithelium cells (HOEC). This study demonstrated that rs2301104 in autophagy pathway gene HIF1A was associated with susceptibility of NSCL/P.

Associations of the microRNA gene polymorphisms with the risk of non-syndromic supernumerary teeth in a Chinese population.

OBJECTIVE: Non-syndromic supernumerary teeth (NSST) are a common type of dental anomaly. microRNAs (miRNAs) play an important role in craniofacial and tooth development. Therefore, we hypothesized that single nucleotide polymorphisms (SNPs) of miRNAs may be associated with the susceptibility of NSST. MATERIALS AND METHODS: Four miRNA SNPs (rs2910164, rs11614913, rs2043556, and rs2682818) were selected, and their associations with NSST susceptibility were evaluated in a case-control study (163 NSST patients and 326 healthy controls). RESULTS: rs2910164 was significantly associated with a risk of lower NSST (additive model: OR = 4.00, 95 % CI = 1.76-9.09, P = 0.001), and rs2682818 showed nominal association with a risk of upper NSST (additive model: OR = 1.40, 95 % CI = 1.02-1.91, P = 0.037) and NSST among male patients (additive model: OR = 1.62, 95 % CI = 1.08-2.43, P = 0.020). CONCLUSION: Genetic variants of miR-146a/rs2910164 and miR-618/rs2682818 were likely associated with the risk of NSST in the Chinese population.

Rs2262251 in lncRNA RP11-462G12.2 is associated with nonsyndromic cleft lip with/without cleft palate.

Nonsyndromic cleft lip with/without cleft palate (NSCL/P) is one of the most common human congenital defects. Rs2262251 (G>C) in long noncoding RNA (lncRNA) RP11-462G12.2 is in high linkage disequilibrium with rs8049367, which was identified in our previous genome-wide association study on NSCL/P, and is a potential causative single-nucleotide polymorphism (SNP) for NSCL/P. To test these hypotheses, rs2262251 was evaluated in another cohort of 1,314 cases and 1,259 controls. Rs2262251 was associated with NSCL/P risk (p = .003). However, no association was detected for cleft palate only. SNP rs2262251 affected the structure and expression of lncRNA RP11-462G12.2 in HEK293 and HEPM cells and in lip tissues from patients with NSCL/P. Overexpression of the rs2262251 G allele contributed to reducing the number of cells in the G0/G1 phase, inhibiting cell apoptosis, and promoting cell proliferation in vitro. The rs2262251 C allele regulated the expression of miR-744-5p and its target gene IQSEC2, both of which were expressed in human lip tissues, and showed reverse correlation during mouse lip development. Taken together, these findings suggest that rs2262251 is associated with the risk of NSCL/P and participates in a lncRNA-miRNA-mRNA regulatory axis in which miR-744-5p and IQSEC2 combine to control NSCL/P development.

Non-syndromic cleft lip with or without palate-susceptible SNPs is associated with hyperdontia.

BACKGROUND: Non-syndromic supernumerary teeth (NSST) or hyperdontia may share common genetic determinants with non-syndromic cleft lip with or without palate (NSCL/P). The aim of this study was to test the associations between five genome-wide-associated NSCL/P-susceptible single nucleotide polymorphisms (SNPs) (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) and the occurrence of NSST. MATERIALS AND METHODS: A total of 163 cases and 326 controls were recruited and their genomic DNA was extracted from blood samples. Five NSCL/P-susceptible SNPs (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) were genotyped by TaqMan method. Odds ratio (OR) and 95% confidence interval (CI) were used to estimate the associations between the SNPs and the risk of NSST by PLINK software. RESULTS: Rs4791774 (A > G) and rs13041247 (T > C) were associated with risk of NSST (rs4791774: Padd  = 0.011, OR, 95% CI = 0.62, 0.43-0.90; rs13041247: Phomo  = 0.031, OR, 95% CI = 1.79, 1.05-3.05) and one supernumerary tooth (rs4791774: Pdom  = 0.009, OR, 95% CI = 0.56, 0.36-0.87; rs13041247: Phomo  = 0.034, OR, 95% CI = 1.82, 1.05-3.15). Rs4791774 (A > G) was also showed association with risk of upper arch supernumerary teeth only (Padd  = 0.010, OR, 95% CI = 0.60, 0.41-0.89). CONCLUSION: Non-syndromic cleft lip with or without palate-susceptible loci rs4791774 (A > G) and rs13041247 (T > C) were associated with the risk of supernumerary teeth.

Non-syndromic cleft lip with or without palate susceptible loci is associated with tooth agenesis.

OBJECTIVE: Non-syndromic tooth agenesis (NSTA) may share common genetic factors with non-syndromic cleft lip with or without cleft palate (NSCL/P). Single-nucleotide polymorphisms (SNPs) were associated with individual's susceptibility to these anomalies. We selected five NSCL/P-associated SNPs from our previous genome-wide association study (GWAS) to test for the associations with NSTA. MATERIALS AND METHODS: A total of 677 NSTA cases and 1,144 healthy controls were recruited in this case-control study. Five genome-wide NSCL/P-associated SNPs (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) were genotyped by TaqMan platform and evaluated for the associations with NSTA using plink software. RESULTS: No significant associations between these SNPs and risk of NSTA were observed in the overall analysis and subgroup analysis with the number of missing teeth. However, in the subgroup analysis by tooth position, rs8049367 was nominally associated with mandibular premolar agenesis (Dominant model: ORdom  = 0.66, 95% CIdom  = 0.47-0.93, pdom  = 0.016; Heterozygote model: ORhet  = 0.60, 95% CIhet  = 0.41-0.88, Phet  = 0.008). Rs4791774 showed a nominal association with congenitally missing maxillary canine (Dominant model: ORdom  = 0.53, 95% CIdom  = 0.28-0.98, pdom  = 0.041; Heterozygote model: ORhet  = 0.50, 95% CIhet  = 0.26-0.97, Phet  = 0.041) and premolar (Additive model: OR = 0.59, 95% CI = 0.36-0.96, p = 0.035). CONCLUSION: This study showed that NSCL/P susceptible loci rs8049367 and rs4791774 were probably associated with the risk of NSTA.