Raihana Zaka

Transforming growth factor-β1 (TGF-β1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression†

Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon‐containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor‐β (TGF‐β), which also regulates FN isoform expression. We have examined the effects of TGF‐β treatment on the assumption of the chondrogenic phenotype in the teratoma‐derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre‐chondrogenic state and, in this state, treated with 10 ng/ml TGF‐β. The cells started to elaborate a matrix rich in sulfated proteoglycans, such that within the first 12 days of culture, TGF‐β1 treatment appeared to slightly accelerate early acquisition of an Alcian blue‐stained matrix, and caused a dose‐ and time‐dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF‐β became indistinguishable from those of the controls. Interestingly, TGF‐β treatment caused a significant dose‐ and time‐dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF‐β induces the early stages of chondrogenic maturation in this pre‐chondrogenic line and that TGF‐β treatment increases expression of FN isoforms that lack the EDA and EDB exons. Published 2005 Wiley‐Liss, Inc.

Role of the progressive ankylosis gene in cartilage mineralization.

Purpose of reviewAmong the myriad of players in the calcification of cartilage, ANK is a relatively new entrant. It is a multipass transmembrane protein that regulates the transport of inorganic pyrophosphate between the cell and the extracellular space. Mutations in ANK result in two distinct calcification disorders: craniometaphyseal dysplasia and familial calcium pyrophosphate dihydrate deposition disease. The purpose of this review is to highlight recent work on the role of ANK in physiological and pathological calcification of articular and growth plate cartilage. Recent findingsNew information on the function of ANK suggests that the protein is part of a constellation of critical components that interact to regulate the elaboration of inorganic pyrophosphate. In addition to ANK, these components include alkaline phosphatase, the ectoenzyme PC-1, and osteopontin. ANK expression is also regulated by a variety of growth factors and cytokines that may further affect the transport of inorganic pyrophosphate and may be particularly relevant to the increased levels of expression of ANK in cartilage from chondrocalcinosis and osteoarthritis patients. SummaryAdditional studies will be required to understand the contribution of ANK in shaping the fine balance of components necessary for crystal deposition in degenerating articular cartilage. Furthermore, the precise role of inherited mutations in ANK on the elaboration of inorganic pyrophosphate, and the ultimate deposition of either basic calcium phosphate or calcium pyrophosphate dihydrate crystals, remains unclear.

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.

Oxygen Tension Regulates the Expression of ANK (Progressive Ankylosis) in an HIF-1-Dependent Manner in Growth Plate Chondrocytes†‡

The proximal promoter region of ANK, a gene that codes for a protein that regulates the transport of inorganic pyrophosphate, contains two hypoxia responsive elements (HREs); therefore, we studied the expression and function of ANK at different oxygen tensions. ATDC5 and N1511 clonal chondrocytic cells were cultured in either hypoxia (2% O2) or normoxia (21% O2). Transcript and protein levels of ANK were depressed in hypoxic conditions, as were levels of extracellular pyrophosphate (ePPi). To determine whether HIF‐1 was involved in the oxemic response, Hif‐1α knockdown cells were exposed to varying oxygen conditions and ANK expression was assessed. Knockdown of Hif‐1α resulted in low levels of expression of ANK in hypoxia and normoxia. Chromatin immunoprecipitation (ChIP) assays explored the binding of Hif‐1α to ANK HREs and showed that Hif‐1α is able to bind to the HREs of ANK more avidly in normoxia than in hypoxia. Furthermore, functional studies of Hif‐1α activity using luciferase reporter assays of wildtype and mutagenized HREs showed that only HRE‐1 binds Hif‐1α in normoxia. Expression of ANK in growth plate and articular cartilage was low in hypoxic regions of the tissues, and higher levels of ANK expression were observed in the synovium and meniscus in regions that have a normally higher oxygen tension. The data suggest that ANK expression and function in vitro and in vivo are repressed in hypoxic environments and that the effect is regulated by HIF‐1.

Phosphate and calcium are required for TGFβ-mediated stimulation of ANK expression and function during chondrogenesis

The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGFβ. The purpose of this study was to determine whether TGFβ stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGFβ increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na+/Pi channels Pit‐1 and Pit‐2, indicated that the stimulation of ANK expression by TGFβ required the influx of phosphate, specifically by the Pit‐1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGFβ on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGFβ. Since previous studies of endochondral ossification in the growth plate have shown that L‐type calcium channels are essential for chondrogenesis, we investigated their role in the TGFβ‐stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L‐type channel Cav1.2 (α1C) inhibited the TGFβ stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGFβ stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation. J. Cell. Physiol. 224: 540–548, 2010.