Interaction of TGFβ and BMP signaling pathways during chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.     

TGFβ‐induced PI 3 kinase‐dependent Mnk‐1 activation is necessary for Ser‐209 phosphorylation of eIF4E and mesangial cell hypertrophy

Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.     

TGFbeta1 regulates endothelial cell spreading and hypertrophy through a Rac-p38-mediated pathway

Background information. TGFβ (transforming growth factor β) is a multifunctional cytokine and a potent regulator of cell growth, migration and differentiation in many cell types. In the vascular system, TGFβ plays crucial roles in vascular remodelling, but the signalling pathways involved remain poorly characterized. Results. Using the model of porcine aortic endothelial cells, we demonstrated that TGFβ stimulates cellular spreading when cells are on collagen I. TGFβ‐stimulated Rac1–GTP accumulation, which was associated with increased MAPK (mitogen‐activated protein kinase) p38 phosphorylation. Furthermore, ectopic expression of a dominant‐negative Rac mutant, or treatment of the cells with the p38 pharmacological inhibitor SB203580, abrogated TGFβ‐induced cell spreading. Our results demonstrate for the first time that prolonged exposure to TGFβ stimulates endothelial cell hypertrophy and flattening. Collectively, these data indicate that TGFβ‐induced cell spreading and increase in cell surface areas occurs via a Rac—p38‐dependent pathway. Conclusions. The Rac—p38 pathway may have conceptual implications in pathophysiological endothelial cell responses to TGFβ, such as wound healing or development of atherosclerotic lesions.     

Fibulin-3 regulates the inhibitory effect of TNF-α on chondrocyte differentiation partially via the TGF-β/Smad3 signaling pathway

Fibulin-3 is an extracellular matrix glycoprotein that is present in elastic tissue and involved in carcinoma development. Previous studies have indicated that fibulin-3 may affect skeletal development, cartilage, and osteoarthritis (OA). This study aims to investigate the function of fibulin-3 on chondrocytes under tumor necrosis factor alpha (TNF-α) stimulation and in murine OA models, and explore the possible mechanism. It was found that fibulin-3 was increased in the cartilage of OA models and in the chondrogenic cells ATDC5 stimulated by TNF-α. Fibulin-3 promoted the proliferation of ATDC5 cells both in the presence and absence of TNF-α. Moreover, overexpression of fibulin-3 suppressed the chondrogenic and hypertrophic differentiation of ATDC5 cells, while knockdown of fibulin-3 caused the opposite effect. Mechanistically, fibulin-3 partially suppressed the activation of TGF-β/Smad3 signaling by inhibiting the phosphorylation of Smad3. SIS3, a Smad3 inhibitor, decreased the chondrogenesis of articular cartilages in OA models, and partially reversed the chondrogenic differentiation of ATDC5 cells caused by knockdown of fibulin-3 in the presence of TNF-α. Furthermore, co-immunoprecipitation (Co-IP) showed that fibulin-3 could only interact with TGF-β type I receptor (TβRI), although overexpression of fibulin-3 reduced the protein levels of both TβRI and TβRII. In conclusion, this study indicates that fibulin-3 modulates the chondrogenic differentiation of ATDC5 cells in inflammation partially via TGF-β/Smad3 signaling pathway.     

Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian‐blue staining, qRT‐PCR, and quantification of alkaline phosphatase (ALP) activity. Pre‐differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)‐β was always required for chondrogenic differentiation and deposition of a collagen‐type‐II‐positive extracellular matrix, while bone morphogenetic protein (BMP)‐2, ‐4, ‐6, ‐7, aFGF, and IGF‐I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF‐β, however, without preventing collagen type X expression. bFGF or parathyroid hormone‐like peptide (PTHrP) inhibited the TGF‐β‐responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up‐regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor‐independent pathway. While TGF‐β was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II⁺/collagen type X^? cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes. J. Cell. Physiol. 223: 84–93, 2010. © 2009 Wiley‐Liss, Inc.     

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.     

Tumour necrosis factor-alpha up-regulates the expression of BMP-4 mRNA but inhibits chondrogenesis in mouse clonal chondrogenic EC cells, ATDC5

Tumour necrosis factor (TNF)-alpha causes the degradation of articular cartilage in arthritis via direct actions on chondrocytes. However, it remains unknown whether TNF-alpha affects chondrogenesis in chondroprogenitors. In the present study, we assessed the effects of TNF-alpha in vitro on chondrogenesis using mouse clonal chondrogenic EC cells, ATDC5. TNF-alpha (10 ng/ml) stimulated [3H] thymidine incorporation in undifferentiated ATDC5 cells, and suppressed cartilaginous nodule formation and the accumulation of cartilage-specific proteoglycan. We recently showed that undifferentiated ATDC5 cells express BMP-4 and that exogenously administered BMP-4 promotes chondrogenesis in these cells. Interestingly, TNF-alpha up-regulated the expression of BMP-4 mRNA in undifferentiated ATDC5 cells in time- and dose-dependent manners. However, exogenously administered BMP-4 was not capable of reversing the inhibitory action of TNF-alpha on chondrogenesis in ATDC5 cells. These results indicate that TNF-alpha stimulates both cell proliferation and BMP-4 expression but inhibits chondrogenesis in chondroprogenitor-like ATDC5 cells.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

Inorganic phosphate modulates responsiveness to 24,25(OH)2D3 in chondrogenic ATDC5 cells

Chondrogenic ATDC5 cells were used as a model of in vitro endochondral maturation to study the role of inorganic phosphate (Pi) in the regulation of growth plate chondrocytes by vitamin D3 metabolites. ATDC5 cells that were cultured for 10 days post‐confluence in differentiation media and then treated for 24 h with Pi produced a type II collagen matrix based on immunohistochemistry and expressed mRNAs for several chondrocytic markers, including aggrecan, collagen types II and X, cartilage oligomeric matrix protein, and SOX9. Pi also caused a decrease in [³⁵S]‐sulfate incorporation and stimulated apoptosis, as evidenced by increased DNA fragmentation and caspase‐3 activity. In addition, treatment with Pi induced sensitivity to 24,25‐dihydroxyvitamin D3 and this effect was both dose‐dependent and was blocked by phosphonoformic acid (PFA), a specific inhibitor of sodium dependent type III Pi transporters. Treatment with 24R,25(OH)₂D₃ reduced cell number and increased alkaline phosphatase specific activity in a dose‐dependent manner. Moreover, 24R,25(OH)₂D₃ reversed the Pi‐induced decrease in incorporation of [³H]‐thymidine and [³⁵S]‐sulfate incorporation, as well as the Pi‐induced increase in apoptosis. These results suggest that Pi acts as an early chondrogenic differentiation factor, inducing response to 24R,25(OH)₂D₃; treatment of committed chondrocytes with Pi induces apoptosis, but 24R,25(OH)₂D₃ mitigates these effects, indicating a possible inhibitory feedback loop. J. Cell. Biochem. 107: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis

Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. However, it remains unclear what causes the differential effects between TGF-β and BMP signaling on prechondrogenic condensation. Our previous report demonstrated that ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation. In addition, TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels. This study suggests that Ca(2+)-driven ATP oscillations mediate TGF-β-induced the initiation step of prechondrogenic condensation and determine the differential effects between TGF-β and BMP signaling on chondrogenesis.     

FLRT2 promotes cellular proliferation and inhibits cell adhesion during chondrogenesis

One of the earliest events during chondrogenesis is the formation of condensations, a necessary pre‐requisite for subsequent differentiation of a chondrogenic phenotype. Members of the Fibronectin Lecucine Rich Transmembrane (FLRT) proteins have been shown to be involved in cell sorting and neurite outgrowth. Additionally, FLRT2 is highly expressed at putative sites of chondrogenic differentiation during craniofacial development. In this study, we demonstrate that FLRT2 plays a role in mediating cell proliferation and cell–cell interactions during early chondrogenesis. Clones of stable transfectants of a murine chondroprogenitor cell line, ATDC5, were established in which FLRT2 was knocked down or overexpressed. Cells in which FLRT2 was knocked down proliferated at a slower rate compared to control wild‐type ATDC5 cells or those containing a non‐coding shRNA. In addition, FLRT2 knockdown cells formed numerous lectin peanut agglutinin (PNA) stained aggregates and exhibited higher expression of the cell adhesion molecule, N‐cadherin. In an in vitro wound healing assay, fewer FLRT2 knockdown cells appeared to migrate into the defect. Surprisingly, the FLRT2 knockdown cells demonstrated increased formation of Alcian blue‐stainable extracellular matrix, suggesting that their reduced aggregate formation did not inhibit subsequent chondrogenic differentiation. The opposite trends were observed in ATDC5 clones that overexpressed FLRT2. Specifically, FLRT overexpressing cells proliferated faster, formed fewer PNA‐positive aggregates, accumulated increased Alcian blue‐positive matrix, and migrated faster to close a wound. Collectively, our findings provide evidence for a role of FLRT2 in enhancing cell proliferation and reducing intercellular adhesion during the early stages of chondrogenesis. J. Cell. Biochem. 112: 3440–3448, 2011. © 2011 Wiley Periodicals, Inc.     

Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation

Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

The importance to chondrocyte differentiation of changes in expression of the multiple inositol polyphosphate phosphatase

It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP(5) and InsP(6). We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates-both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP(6) levels in ATDC5 cells. However, InsP(5) levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP(5) and InsP(6) levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP(5) and InsP(6). Thus, substantial changes in MINPP expression can occur without a net effect upon InsP(5) and InsP(6) turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.