Differentiation Of ATDC5 Cells Research Articles

Synthetic poly(2-acrylamido-2-methylpropanesulfonic acid) gel induces chondrogenic differentiation of ATDC5 cells via a novel protein reservoir function.

We previously demonstrated that a synthetic negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induced chondrogenic differentiation of ATDC5 cells. In this study, we clarified the underlying molecular mechanism, in particular, focusing on the events that occurred at the interface between the gel and the cells. Gene expression profiling revealed that the expression of extracellular components was enhanced in the ATDC5 cells that were cultured on the PAMPS gel, suggesting that extracellular proteins secreted from the ATDC5 cells might be adsorbed in the PAMPS gel, thereby contributing to the induction of chondrogenic differentiation. Therefore, we created "Treated-PAMPS gel," which adsorbed various proteins secreted from the cultured ATDC5 cells during 7 days. Proteomic analysis identified 27 proteins, including extracellular matrix proteins such as Types I, III, and V collagens and thrombospondin (THBS) in the Treated-PAMPS gel. The Treated-PAMPS gel preferentially induced expression of chondrogenic markers, namely, aggrecan and Type II collagen, in the ATDC5 cells compared with the untreated PAMPS gel. Addition of recombinant THBS1 to the ATDC5 cells significantly enhanced the PAMPS-induced chondrogenic differentiation, whereas knockdown of THBS1 completely abolished this response. In conclusion, we demonstrated that the PAMPS gel has the potential to induce chondrogenic differentiation through novel reservoir functions, and the adsorbed THBS plays a significant role in the induction.

Enhanced chondrogenesis in a coculture system with genetically manipulated dedifferentiated chondrocytes and ATDC5 cells.

Articular cartilage repair after injury is a great challenge worldwide due to its nerveless and avascular features. Tissue engineering is proposed as a promising alternative for cartilage regeneration. In this study, an adenoviral vector carrying the transforming growth factor-β3 (TGF-β3) gene was constructed and introduced into dedifferentiated chondrocytes, which were then cocultured with ATDC5 cells in an alginate hydrogel system. The results showed that the experimental groups exhibited better cell viability and higher levels of cartilage-related genes than the control groups. In this coculture system, the chondrogenic differentiation of ATDC5 cells was effectively induced by TGF-β3 and other latent cytokines that were produced by the transfected chondrocytes. Thus, this method can avoid the degradation of exogenous TGF-β3, and it can protect ATDC5 cells during virus transfection to maintain cell viability and chondrogenic differentiation capability. Taken together, this study provides fresh insights for applying this genetically manipulated coculture system to cartilage repair in the future.

Regulation of the Viscoelastic Properties of Hyaluronate-Alginate Hybrid Hydrogel as an Injectable for Chondrocyte Delivery.

Modulation of the viscoelastic properties of hydrogels is critical in tissue engineering applications. In the present study, a hyaluronate–alginate hybrid (HAH) was synthesized by introducing alginate to the hyaluronate backbone with varying molecular weights (700–2500 kDa), and HAH hydrogels were prepared in the presence of calcium ions at the same cross-linking density. The storage shear moduli of the HAH hydrogels increased with the concomitant increase in the molecular weight of hyaluronate in the HAH polymer. The HAH hydrogels were also modified with arginine–glycine–aspartic acid (RGD) and histidine–alanine–valine (HAV) peptides to enhance cell–matrix and cell–cell interactions, respectively. The chondrogenic differentiation of ATDC5 cells encapsulated within the HAH hydrogels was enhanced with the increase in the storage shear moduli of the gels in vitro as well as in vivo. This approach of regulating the viscoelastic properties of hydrogels using polymers of varying molecular weights at the same cross-linking density may prove to be useful in various tissue engineering applications including cartilage regeneration.

Integrin α4 mediates ATDC5 cell adhesion to negatively charged synthetic polymer hydrogel leading to chondrogenic differentiation

Negatively charged synthetic hydrogels have been known to facilitate various cellular responses including cell adhesion, proliferation, and differentiation; however, the molecular mechanism of hydrogel-dependent control of cell behavior remains unclear. Recently, we reported that negatively charged poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel induces chondrogenic differentiation of ATDC5 cells via novel protein reservoir function. In this study, we identified the cell adhesion molecules binding to PAMPS gels that act as mechanoreceptors. First, we performed a pull-down assay by particle gels using cell membrane proteins of ATDC5, and found that multiple membrane proteins bound to the PAMPS gel, whereas the uncharged poly(N,N′-dimethylacrylamide) gel as control did not bind to any membrane proteins. Western blot analysis indicated differential binding of integrin (ITG) isoforms to the PAMPS gel, in which the α4 isoform, but not α5 and αv, efficiently bound to the PAMPS gel. ITG α4 knockdown decreased cell spreading of ATDC5 on PAMPS gels, whereas the enhanced expression increased the behavior. Furthermore, ITG α4 depletion suppressed PAMPS gel-induced expression of bone morphogenic protein (BMP) 4 contributing to chondrogenic differentiation, in concordance with the reduction of ERK activation. These results demonstrated that membrane protein binding to PAMPS gels occurred in a charge-dependent manner, and that ITG α4 plays a crucial role in cell spreading on PAMPS gels and acts as a mechanoreceptor triggering cellular signaling leads to chondrogenic differentiation.

Regulation of chondrocyte hypertrophy in an osteochondral interface mimicking gel matrix

Calcified cartilage extracellular matrix (ECM) is a critical interface at the osteochondral junction which plays an important role in maintaining the structural continuity between articular cartilage and subchondral bone. This mineralized network is primarily composed of glycosaminoglycans (GAGs) and collagen type II (col II) and hosts hypertrophic chondrocytes. This work aimed to investigate the effect of gel composition and collagen II content on the behavior and hypertrophic differentiation of ATDC5 cells for regeneration of calcified cartilage tissue. For this purpose, chitosan/collagen type II/nanohydroxyapatite (chi/col II/nHA) composite hydrogels were prepared to mimic the calcified cartilage ECM. ATDC5 cells were encapsulated within the composite gels and the viability, ECM production and hypertrophic gene expression were assessed during culture. All composites were favorable for ATDC5 viability and proliferation, whereas specific ECM production and hypertrophic differentiation were dependent on gel composition. Chitosan: collagen II ratio had an impact on ATDC5 cell fate. Hypertrophic differentiation was best pronounced in chi/col II/nHA 70:30 composition. The results obtained from this study offers a scaffold-based approach for calcified cartilage regeneration and provide an insight for biomimetic design and preparation of more complicated gradient osteochondral units.

Deletion of p21 expression accelerates cartilage tissue repair via chondrocyte proliferation

Articular cartilage tissue has a poor healing potential, and when subjected to traumatic damage this tissue undergoes cartilage degeneration and osteoarthritis. The association between the regulation of cell cycle checkpoints and tissue regeneration has been previously investigated, and p21 was initially identified as a potent inhibitor of cell cycle progression. However, the effects of p21 defects on damaged tissue remain controversial. Therefore, the aim of the present study was to evaluate the effects of p21 deficiency on cartilage repair. A mouse model of articular cartilage repair was generated by inducing a patellar groove scratch in 8‑week‑old p21‑knockout (KO) mice and C57Bl/6 wild‑type (WT) mice. Mice were sacrificed at 4and8weeks post‑surgery. The present study also investigated the effect of p21 deficiency on cartilage differentiation in ATDC5 cells invitro. Safranin O staining results indicated that cartilage repair initially occurred in p21 KO mice. In addition, immunohistochemical analysis demonstrated that p21 KO upregulated proliferating cell nuclear antigen and increased cell proliferation. However, typeII collagen and Sox9 expression levels remained unchanged in p21 KO and WT mice. Moreover, it was identified that p21 downregulation did not affect Sox9 and typeII collagen expression levels invitro. Furthermore, p21 deficiency promoted healing of articular cartilage damage, which was associated with cell proliferation invivo, and increased chondrocyte proliferation but not differentiation invitro. Therefore, the present results suggested that p21 does not affect Sox9 or typeII collagen expression levels during cartilage differentiation in the repair of cartilage defects.

Neuropeptide Y enhances proliferation and chondrogenic differentiation of ATDC5 cells

In recent years, emerging evidence has illustrated the indispensable role of sympathetic neurotransmitters and their receptors in cartilage mediation. The presence of neuropeptide Y (NPY)-positive sympathetic nerve fibres in cartilage and NPY-secretion function in chondrocytes raises the possibility of NPY directly regulating the function of chondrocytes. Therefore, this study intended to evaluate the effect of NPY and its receptors on the proliferation and chondrogenic differentiation of ATDC5 cells. Results showed NPY, especially at a concentration of 10−10 M, to significantly enhance proliferation of ATDC5 cells. Moreover, NPY effectively facilitated early chondrogenesis and late hypertrophy/mineralisation of ATDC5 cells via Y1 receptor signalling, rather than via Y2 receptor signalling. Taken together, the results help us to understand how NPY and its receptors affect the function of chondrocytes.

An oriented hydroxyapatite film with arrayed plate-like particles enhance chondrogenic differentiation of ATDC5 cells.

Osteochondral defects of articular cartilage cannot regenerate spontaneously. For its surgical treatment, advancements in cartilage tissue engineering have particularly focused on subchondral bone lesions that tend to delay healing. Therefore, it is important to understand interactions between subchondral bone and chondrocytes. This study aimed to investigate the behavior of chondrogenic ATDC5 cells on oriented hydroxyapatite (HAp) films that mimic bone surfaces. HAp nanoparticles prepared herein were needle like and plate like. HAp films were formed through self-organization of the nanoparticles and had 2D structures regularly arrayed with the particles. Both films prominently comprised a-plane orientation surfaces but differed in the degree of hydrophilicity because of the patterns of particle self-assembly. ATDC5 cells cultured on the HAp film with plate-like particles could adhered in a shorter period but could not spread. The adhesive force of cells was weaker with the hydrophilic surface than with other surfaces, as determined using a trypsin-based cell detachment assay. In addition, ATDC5 cells displayed enhanced proliferation and chondrogenic differentiation. Our results suggest that the oriented HAp film formed using plate-like particles provided chondrogenic cells with a desired scaffold as that of subchondral bone to increase cell proliferation and differentiation.

Plate-like hydroxyapatite synthesized from dodecanedioic acid enhances chondrogenic cell proliferation.

The scaffold for head and neck reconstruction needs mechanical strength to maintain specific forms. Hydroxyapatite (HA) enhances the mechanical strength of hydrogel and is routinely used for cartilage regeneration. However, there is a demand for hydroxyapatite that controls chondrogenic cell behavior. Our aim was to regulate HA morphology through a hydrothermal process using organic acid and enhance chondrocyte proliferation and differentiation using shaped-regulated HA. HA was synthesized from dodecanedioic acid (DD:HA) and oleic acid (OA:HA) by a hydrothermal method and then coated onto glass plates. Surface properties of the samples were compared by various techniques. Surface roughness and contact angles were calculated. Proliferation and differentiation of chondrogenic cells were measured by MTT assays and Alcian Blue staining, respectively, after various incubation periods. The morphological structures of DD:HA and OA:HA were different; however, the crystallinity and chemical structures were similar. Surface roughness and hydrophilic behavior were higher on DD:HA. DD:HA enhanced chondrogenic cell proliferation over time. The differentiation of ATDC5 cells was also increased on the DD:HA surface compared with those in other groups. DD:HA enhanced cell viability to a greater extent than OA:HA did, indicating its excellent potential as an inorganic material compatible with chondrocyte regeneration.

MiR-211-5p contributes to chondrocyte differentiation by suppressing Fibulin-4 expression to play a role in osteoarthritis.

MicroRNAs (miRNAs) serve as key regulators in human disorders. Previous research reported that miR-211-5p is down-regulated in osteoarthritis (OA) and that Fibulin-4 inhibits chondrocyte differentiation. However, the role of miR-211-5p in the development of OA has not been clarified, and its downstream target has not been studied. This study aimed to explore the effect of miR-211-5p on chondrocyte differentiation and its influence on OA pathogenesis, as well as the interaction between miR-211-5p and Fibulin-4. In this study, we found that miR-211-5p is significantly down-regulated in articular cartilage tissues in an OA rat model, whereas it is clearly up-regulated during chondrocyte differentiation of ATDC5 cells. Silencing miR-211-5p in ATDC5 cells had an adverse effect on chondrocyte differentiation. Fibulin-4 was identified as a target of miR-211-5p, and miR-211-5p participated in chondrocyte differentiation by negatively regulating Fibulin-4 expression. In the OA rat model, miR-211-5p overexpression facilitated chondrocyte differentiation, along with the reduced pro-inflammatory cytokines level and the level of proteinases responsible for cartilage matrix degradation. In summary, miR-211-5p promotes chondrocyte differentiation by negatively regulating Fibulin-4 expression, and represses the expression of pro-inflammatory cytokines and proteinases responsible for cartilage matrix degradation in OA. miR-211-5p may serve as a promising target for OA treatment.

MiR-20a suppresses chondrogenic differentiation of ATDC5 cells by regulating Atg7

Both the miR-17-92 cluster and autophagy have been suggested as critical regulators of bone development, but the potential correlation between the two factors is largely unknown. Hence, we investigated whether members of this cluster can regulate chondrogenesis through an autophagy-related signalling pathway. In this study, the expression of miR-17-92 cluster members and the level of autophagic activity were investigated during chondrogenic induction in ATDC5 cells. miR-17, miR-18a, miR-20a, and miR-92-1 showed significant changes, and the level of autophagic activity was enhanced. Among the miR-17-92 cluster members, miR-20a showed the most significant change. Histological, cellular and molecular analyses were performed after the regulation of miR-20a and autophagy. miR-20a and autophagy had the opposite effect on chondrogenic differentiation, and there was a negative correlation between them. Moreover, the expression of the autophagy regulatory gene Atg7 was inhibited by miR-20a. siRNA was then used to knock down Atg7, and the results further indicated that Atg7 might be a potential target of miR-20a in chondrogenic differentiation. In conclusion, miR-20a is a critical negative regulator of chondrogenic differentiation because it inhibits autophagy via Atg7. Other members of the miR-17-92 cluster may have a similar effect, but this hypothesis requires further investigation.

Hyaluronate-alginate hybrid hydrogels prepared with various linkers for chondrocyte encapsulation

Tissue engineering typically requires a use of scaffolds when delivering tissue-specific cells to be engineered. Hydrogels are frequently used as scaffolds, because their composition, structure, and function resemble the natural tissue extracellular matrix. In this study, hyaluronate-alginate hybrid (HAH) was synthesized by conjugating alginate (ALG) with the hyaluronate (HA) backbone using various types of linkers. HAH hydrogel was prepared by physically cross-linking the HAH polymer in the presence of calcium ions without chemical cross-linkers. The mechanical stiffness of HAH hydrogel was significantly affected by changing the type of a linker between HA and ALG. The mechanical stiffness increased with increasing linker length, likely due to enhanced intermolecular reactions between HA and ALG. This enables controlling the mechanical properties of HAH hydrogels. The types of linkers used to synthesize HAHs also influenced the chondrogenic differentiation of ATDC5 cells cultured in HAH hydrogel in vitro. This hybrid system that can change the mechanical stiffness by varying the linker type while maintaining the cross-linking density may be useful to design and fabricate scaffolds for tissue engineering applications, including cartilage regeneration.

Nutritional supplementation with myo-inositol in growing mice specifically augments mandibular endochondral growth.

The purpose of this study was to examine growth-promoting effects of myo-inositol nutritional supplementation on the mandible in experimental animals. Mice were fed on diets that contained various concentration of myo-inositol for 3 to 12 weeks. The length of the mandible, maxilla, and femur were measured on μCT images. The mandible and tibia were examined histologically and immunohistochemically. The effects of myo-inositol on cell proliferation and chondrocytic differentiation were examined using ATDC5 cells. Myo-inositol supplementation had no effects on body weight, length, and maxilla and femur lengths. However, the length of mandible and the thickness of the mandibular condylar cartilage (MCC) were increased by myo-inositol supplement. Microarray analysis revealed that Pik3cd was highly expressed in MCC as compared to that in the cartilage of the tibial growth plate, which was confirmed by real-time RT-PCR and immunohistochemistry. ATDC5 cells also highly expressed Pik3CD. Myoinositol induced increases in cell proliferation and chondrocytic differentiation in ATDC5 cells. The addition of a PIK3CD inhibitor blocked the induction of cell proliferation by myo-inositol in ATDC5 cells. Nutritional supplementation with myo-inositol in growing mice augmented mandibular endochondral growth without any systemic effects. The specific promotion of mandibular growth by myoinositol is primarily dependent on the specific intensive expression of PIK3CD in the MCC.

Ran, Ras-related nuclear protein, regulates chondrogenic differentiation of ATDC5 cells

Ran, Ras-related nuclear protein, regulates chondrogenic differentiation of ATDC5 cells

Expression of Halo-hFGF18 and study of its effect on differentiation of ATDC5 cells

Fibroblast growth factor 18 (FGF18) is a member of the fibroblast growth factor family and important in cartilage growth and development. However, the mechanism by which FGF18 mediates its biological functions is still unclear. In our study, we expressed the rhFGF18 protein fused to a HaloTag, (Halo-rhFGF18). MTT assay results indicated that both rhFGF18 and Halo-rhFGF18 have similar biological activities in NIH3T3 cells. However, basic FGF and acidic FGF were more potent than both rhFGF18 and Halo-rhFGF18. Confocal imaging data indicated that the red fluorescence labeled Halo-rhFGF18 strongly bound to ATDC5 cells and stimulated their proliferation and differentiation, which suggests that glycosaminoglycans may be involved in mediating the biological effects of rhFGF18 in ATDC5 cells. Moreover, western blot results demonstrated that, in ATDC5 cells, ERK1/2 signaling is activated upon stimulation with rhFGF18. Our results may open doors for the use of rhFGF18 as a drug to promote cartilage growth.

The osteoarthritis-associated gene PAPSS2 promotes differentiation and matrix formation in ATDC5 chondrogenic cells.

3′-Phosphoadenosine 5′-phosphosulfate synthetase 2 (PAPSS2) has been shown to be important in the development of normal skeletal structure. The aim of the present study was to evaluate the role of PAPSS2 in the differentiation of chondrocytes as well as their mechanisms. Using RNA interference-mediated via a lentivirus and a retrovirus, PAPSS2 gene silence and overexpression in ATDC5 chondrogenic cells were performed. Chondrocyte differentiation and chondrogenic-related gene markers associated with extracellular matrix formation were noted. The mRNA and protein expression for Wnt4, β-catenin and SOX9 genes were observed. The PAPSS2 transcript expression levels progressively decline in ATDC5-induced chondrocyte-like cells during differentiation. Silencing of PAPSS2 expression had a significantly attenuating effect on cell differentiation and decreased expression of collagen II and X. In contrast, over-expression of PAPSS2 promoted the differentiation of ATDC5 chondrogenic cells. The mRNA expression levels of Wnt4 and SOX9 decreased significantly in PAPSS2 knock down cells vs. control cells. However, this expression was increased in the cells over-expressing PAPSS2. These data indicate that PAPSS2 regulates aggrecan activity as well as cell differentiation. The findings favor a mechanism by which PAPSS2 induces differentiation in ATDC5 cells via direct regulation of early signaling events that promote formation of collagenous matrix components. This control is probably mediated via extracellular matrix formation Wnt/β-catenin signaling pathways.

The hsa-miR-181a-5p reduces oxidation resistance by controlling SECISBP2 in osteoarthritis

BackgroundThe phenotypes of osteoarthritis (OA) consist of cartilage extracellular matrix (ECM) metabolism disorder and the breakdown of cartilage homeostasis, which are induced by pro-inflammatory factors and oxidative stress. Selenoproteins regulated by selenocysteine insertion sequence binding protein 2 (SBP2) are highly effective antioxidants, but their regulatory mechanisms, particularly the involvement of miRNAs, are not fully understood.MethodsTo explore whether miR-181a-5p and SBP2 are involved in OA pathogenesis, we established an IL-1β model using the chondrocyte SW1353 cell line. Next, we up- or down-regulated SBP2 and miRNA-181a-5p expression in the cells. Finally, we measured the expression of miRNA-181a-5p, SBP2 and three selenoproteins in OA cartilage and peripheral blood.ResultsThe results showed that IL-1β increased hsa-miR-181a-5p and decreased SBP2 in a time- and dose-dependent manner. GPX1 and GPX4, which encode crucial glutathione peroxidase antioxidant enzymes, were up-regulated along with SBP2 and miR-181a-5p. Furthermore, SBP2 showed a significant negative correlation with miR-181a-5p during induced ATDC5 cell differentiation. There was lower GPX1 and GPX4 mRNA expression and SBP2 protein expression in damaged cartilage than in smooth cartilage from the same OA sample, and hsa-miR-181a-5p expression on the contrary. Similar results were observed in peripheral blood. In conclusion, we have reported a novel pathway in which pro-inflammatory factors, miRNA, SBP2 and selenoproteins are associated with oxidation resistance in cartilage.ConclusionOverall, this study provides the first comprehensive evidence that pro-inflammatory factors cause changes in the cartilage antioxidant network and describes the discovery of novel mediators of cartilage oxidative stress and OA pathophysiology. Our data suggest that miR-181a-5p may be used to develop novel early-stage diagnostic and therapeutic strategies for OA.

The effect of lentivirus-mediated SIRT1 gene knockdown in the ATDC5 cell line via inhibition of the Wnt signaling pathway

SIRT1 is a highly conserved type III acetyltransferase gene located on chromosome 10 in mammals that belong to the Sirtuins family. In order to explore the effects of the SIRT1 gene in the ATDC5 cell line, an RNAi SIRT1 target sequence was designed and synthesized, aimed to knockdown the expression of SIRT1 in ATDC5 by a lentivirus. Gene chip, qrt-PCR, and WES analyses were used to detect the expression of SIRT1 and changes to the Wnt signaling pathway, while detecting any changes in proliferation and differentiation factors. The results showed that the expressions of the SIRT1 gene, mRNA, and protein were lower after transfection of the RNAi SIRT1sequence into ATDC5 cells. The Wnt signaling pathway, especially the classical pathway, was inhibited by the knockdown of SIRT1. The cartilaginous proliferation and differentiation of ATDC5 cells were simultaneously inhibited, and apoptosis was accelerated. In summary, knocking down SIRT1 gene increased the degeneration of ATDC5 cells via inhibiting the Wnt signaling pathway. We also found some novel factors related to the Wnt signaling pathway after SIRT1 gene knockdown (BIRC3, IL1RAP, PPP3CA, PPP2R2A, PPP2R5E, GSN, PPP2R1B, etc), which might provide new clues in disease research related to chondrocyte degeneration.

Novel Bionanocellulose/κ-Carrageenan Composites for Tissue Engineering

In this work, novel bacterial cellulose/κ-carrageenan (BNC/κ-Car) composites, being potential scaffolds for tissue engineering (TE), and outperforming the two polymers when used as scaffolds separately, were for the first time obtained using an in situ method, based on the stationary culture of bacteria Komagateibacter xylinus E25. The composites were compared with native BNC in terms of the morphology of fibers, chemical composition, crystallinity, tensile and compression strength, water holding capacity, water retention ratio and swelling properties. Murine chondrogenic ATDC5 cells were applied to assess the utility of the BNC/κ-Car composites as potential scaffolds. The impact of the composites on the cells viability, chondrogenic differentiation, and expression patterns of Col1α1, Col2α1, Runx2, and Sox9, which are indicative of ATDC5 chondrogenic differentiation, was determined. None of the composites obtained in this study caused the chondrocyte hypertrophy. All of them supported the differentiation of ATDC5 cells to more chondrogenic phenotype.

Treacher Collins syndrome 3 (TCS3)-associated POLR1C mutants are localized in the lysosome and inhibits chondrogenic differentiation

Treacher Collins syndrome (TCS) is a craniofacial developmental disorder whose key feature is a combination of symptoms. For example, a patient could have bilateral downward slanting of the palpebral fissures, colobomas of the lower eyelids, hypoplasia of the facial bones, cleft palate, malformation of the external ears, and atresia of the external auditory canals. TCS3 is caused by mutations of the polr1c gene, which encodes RNA polymerase I and III subunit C (POLR1C). There have been two known missense mutations (Arg279-to-Gln [R279Q] and Arg279-to-Trp [R279W]) at the Arg-279 position. However, it remains to be clarified whether or how both or each individual mutation affects the cellular properties of POLR1C. Here we show that TCS3-associated missense mutations cause aberrant intracellular localization of POLR1C, inhibiting chondrogenic differentiation. The wild type POLR1C is normally localized in the nuclei. The R279Q or R279W mutant is primarily found to be localized in the lysosome. Expression of the R279Q or R279W mutant in mouse chondrogenic ATDC5 cells decreases phosphorylation of 4E-BP1 and ribosomal S6 proteins, which belong to the mammalian target of rapamycin (mTOR) signaling involved in critical roles in the lysosome. Furthermore, expression of the R279Q or R279W mutant inhibits chondrogenic differentiation in ATDC5 cells. Taken together, TCS3-associated mutation leads to the localization of POLR1C into the lysosome and inhibits chondrogenic differentiation, possibly explaining a portion of the pathological molecular basis underlying Treacher Collins syndrome.