Chondrocytes, derived from a tissue that remains as permanent hyaline cartilage in vivo (embryonic chicken caudal sterna) were treated with 10(-8) to 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These nonadherent rounded chondrocytes acquired an adherent, polygonal morphology in a dose-dependent fashion with 1,25(OH)2D3 treatment. During the first 4 days of 1,25(OH)2D3 treatment cell flattening was associated with a 10-fold increase in beta-actin and fibronectin and their corresponding messenger RNAs (mRNAs). After adherence over the 12 days of continuous hormone treatment, a 2- to 4-fold increase in DNA synthesis and DNA accumulation were observed for the highest hormone dose (10(-8) M). Over the same time course total collagen synthesis decreased 35-50% primarily due to decreased type II collagen synthesis, which accompanied comparable decreases in its mRNA. In contrast, both alpha 1(I) and alpha 2(I) showed a continuous 5- to 10-fold increase; however, type I collagen protein synthesis remained undetectable, indicating translational control of the type I collagen synthesis. alpha 1(X) mRNAs showed a 2- 3-fold increase after 12 days of hormone treatment, and its polypeptide was clearly detected by sodium dodecyl sulfate polyacrylamide gel analysis. Type IX collagen synthesis showed a 2-fold increase in synthesis and its mRNA levels during the first 4 days of 1,25(OH)2D3 treatment but thereafter had levels comparable to control cultures. Analysis of proteoglycan synthesis and core protein mRNA levels showed there was a 2-fold increase in core protein mRNAs while proteoglycan synthesis, as assessed by 35S incorporation, showed only a 10-20% increase. Direct hormone effects vs. those secondary to altered cellular morphology were determined by blocking cell adherence by growth of the 1,25(OH)2D3-treated cultures on bacteriological petri dishes. All of the observed effects on cytoskeletal and collagen mRNAs were blocked except the elevations observed in proteoglycan core protein and alpha 1(IX) mRNAs. DNA contents in hormone-treated cultures also remained elevated. These results suggest that 1,25(OH)2D3 both activates and suppresses specific genes, promoting chondrocyte maturation toward a more hypertrophic phenotype. However, prevention of the initial morphological alterations that are induced by 1,25(OH)2D3 blocks many of the subsequent changes in connective tissue expression.

An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in 953 nucleotide segment of DNA consisting of 4 exons and 3 introns. Although the introns in the rat gene are larger, its overall organization is similar to the human gene. Analysis of the 5' flanking sequences of the rat gene shows a modular organization of the promotor as reflected by the the presence of at least 3 classes of regulatory elements. These include (1) typical sequences associated with most genes transcribed by RNA polymerase II (e.g. TATA, CAAT, AP1, AP2), (2) a series of consensus sequences for cyclic nucleotide responsive elements and several hormone receptor binding-sites (estrogen, thyroid and clusters of AG-rich putative Vitamin D responsive elements); and (3) a 24 nucleotide highly conserved sequence between the rat and human gene having a CAAT motif as a central element, designated as an "osteocalcin box." Two regulatory factors of osteocalcin gene expression have been identified. First, contained within the 600 nucleotides immediately upstream from the transcription initiation site are sequences which support Vitamin D dependent transcription of the rat osteocalcin gene. 1,25(OH)2D3 increases osteocalcin mRNA by 6-20 fold increases. In contrast, up to a 200 fold increase in osteocalcin gene expression occurs with mineralization of the extracellular matrix produced by osteoblasts. We propose osteocalcin is a bone-specific marker protein of the mature osteoblast in a mineralizing matrix.

A comparative study of Type X collagen expression, chondrocyte morphology, and the expression of two genes controlling chondrocyte morphology (beta-actin and fibronectin) was carried out on chondrocytes derived from a tissue that remains as permanent hyaline cartilage in vivo (embryonic chicken caudal sterna), from a tissue that undergoes endochondral replacement (embryonic chicken ventral vertebrae) and caudal sternal chondrocytes treated with 1,25(OH)2D3. Under identical in vitro growth conditions and times, sternal chondrocytes grew as rounded non-adherent cells, and vertebral chondrocytes grew as adherent polygonal cells. Upon treatment with 10(-8) M 1,25(OH)2D3 over a twelve day period the sternal chondrocytes showed complete adherence and took on an identical appearance as the vertebral chondrocytes. Cellular adherence of both vertebral and 1,25(OH)2D3 treated sternal chondrocytes was associated with 10 X increased beta-actin, fibronectin and their corresponding mRNA's. Changes in connective tissue expression were observed with altered cellular morphology. Total collagen synthesis was 35-50% lower in both hormone treated and vertebral chondrocytes. Type II collagen was the major collagen type produced by all chondrocyte cultures; however, in both vertebral and 1,25(OH)2D3 treated sternal chondrocytes, a 60 kD collagenous protein was identified. This short chain collagen was determined to be Type X collagen based on its molecular weight and its CNBr peptide maps. Analysis of Type X mRNA levels using a 33 base pair anti sense oligonucleotide sequence to Type X, demonstrated that vertebral cells showed six to seven times more mRNA than sternal chondrocytes. However, the low mRNA levels of type X mRNA in sternal chondrocytes were increased two to three times by 1,25(OH)2D3 treatment. These studies demonstrate that the steroid hormone 1,25(OH)2 vitamin D3 induced morphological, biochemical and molecular changes indicative of chondrocyte maturation from a hyaline to a more hypertrophic phenotype.