Annexin-mediated Ca2+ Influx Regulates Growth Plate Chondrocyte Maturation and Apoptosis

Abstract

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca 2؉ influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca 2؉ , whereas K-201, a specific annexin Ca 2؉ channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca 2؉ influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Maturation of epiphyseal growth plate chondrocytes, which plays an important role during endochondral ossification, is accompanied by major changes of chondrocyte morphology, biosynthetic activities, and energy metabolism. These processes involve an ordered progression of various cell differentiation stages, including proliferation, hypertrophic differentiation, terminal differentiation, and ultimately programmed cell death (apoptosis) (1, 2). During normal development these sequential events are under the strict control of local and systematic factors such as hormones and growth factors. If these processes, however, occur during pathological conditions, they can result in serious cartilage or bone defects. Evidence of endochondral ossification is also seen during osteophyte formation in osteoarthritic cartilage (3, 4). Terminal differentiation of growth plate chondrocytes is an essential process, which primes the cartilage skeleton for its subsequent invasion by osteoblasts and its replacement by a bone matrix. Despite the obvious importance of these terminal differentiation events still little is known about mechanisms regulating these processes.cbfa1, a member of the runt domain family of transcription factors, was originally discovered as a key transcription factor, which controls osteoblast differentiation. In cbfa1-null mice no endochondral and intramembranous bone formation occurs due to an arrest in osteoblast differentiation (5-8). Recent studies have indicated that cbfa1 also plays an important regulative role in terminal chondrocyte maturation. Transgenic mice, which overexpress cbfa1 in non-hypertrophic chondrocytes, display an acceleration of endochondral ossification. Overexpression of cbfa1 in chondrocytes of cbfa1-null mice partially rescued the abnormalities of cbfa1-null mutant mice. In particular, it rescued hypertrophic chondrocyte differentiation in the humerus and femur (9). Thus, cbfa1 seems to play dual functions in endochondral bone formation; it plays a key role in osteoblast differentiation from mesenchymal precursor cells, and it has the ability to stimulate hypertrophic and terminal chondrocyte differentiation.Chondrocyte hypertrophy and terminal differentiation are accompanied by an increase ...

Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells

“…24,26,27 Activation of apoptotic endonucleases 28 eliciting DNA cleavage and chromatin condensation has been well documented. 29,30,31 A tight buffering of intracellular Ca 2 þ is required for normal growth. In fact, apoptosis can be induced by Ca 2 þ mobilization from intracellular pools, 23,24,27 chelation of intracellular Ca 2 þ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N 1 ,N 1 -tetra-acetic acid tetra-acetoxymethyl ester (BAPTA-AM) 28,29 or removal of extracellular Ca 2 þ .…”

“…29,30,31 A tight buffering of intracellular Ca 2 þ is required for normal growth. In fact, apoptosis can be induced by Ca 2 þ mobilization from intracellular pools, 23,24,27 chelation of intracellular Ca 2 þ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N 1 ,N 1 -tetra-acetic acid tetra-acetoxymethyl ester (BAPTA-AM) 28,29 or removal of extracellular Ca 2 þ . 32 Intracellular Ca 2 þ deficiency regulates gene expression 22,26 and induces apoptosis through caspases activation.…”

“…To assess whether intracellular Ca 2 þ depletion would per se cause apoptosis, we cultured PNT1A cells in the presence of BAPTA-AM, a well-known intracellular Ca 2 þ blocker 26 that can trigger activation of caspases and apoptosis. 23,28,29,33 Cell proliferation was assessed by crystal violet staining method in the presence of 20 mM BAPTA-AM. As soon as 4 h after treatment, cells growth was significantly inhibited reaching about 55 and 78% inhibition at 24 and 48 h (Po0.01, data not shown), respectively, in strict association with chromatin condensation and fragmentation, cell rounding and detachment, hallmarks of anoikis (see Figure 1e, RPMI þ BAPTA-AM).…”

Mixing model systems: Using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development

“…In the higher extracellular Ca 2+ , the annexins bind phosphatidylserine in a calcium-dependent manner and act as Ca 2+ channels, allowing an influx of Ca 2+ into the matrix vesicle. Interactions with Type II and Type X collagen stimulate the activity of annexin V by 2-3-fold (Kirsch et al, 2000;Wang et al, 2003), allowing close regulation of the site of nucleation in the extracellular matrix (Fig. 2D).…”

“…The activity of annexin family members are crucial for initiating increases in intravesicular Ca 2+ within the matrix vesicles of developing bone only when the vesicle is in contact with the correct extracellular matrix (collagens II and X) (Anderson, 2003;Kirsch et al, 2000;Wang et al, 2003). Studies of annexin homologues in the zebrafish have not identified expression of these proteins in the inner ear-despite whole-mount in situ hybridization at the appropriate time points (Farber et al, 2003).…”