Eleanor B. Golden

Retinoic acid treatment induces type x collagen gene expression in cultured chick chondrocytes

The vitamin A derivative retinoic acid (RA) is widely thought to be involved in cartilage development, but its precise roles and mechanisms of action in this complex process remain unclear. We have tested the hypothesis that RA is involved in chondrocyte maturation during endochondral ossification and, in particular, is an inducer of maturation-associated traits such as type X collagen and alkaline phosphatase. Immature chondrocytes isolated from the caudal region of Day 19 chick embryo sterna were seeded in secondary monolayer cultures and treated either with a high dose (100 nM) or with physiological doses (10-35 nM) of RA for up to 3 days. We found that after an initial lag of about 24 h, physiological doses of RA indeed induced type X collagen gene expression in the immature cells. This induction was not accompanied by obvious changes in expression of the type II collagen and large aggregating proteoglycan core protein genes. As revealed by immunocytochemistry, 30-35% of the cells in cultures treated with RA for 3 days were engaged in type X collagen production. Interestingly, these cells were relatively similar in size to chondrocytes in which no type X collagen was detected, suggesting that chondrocytes can initiate type X collagen production independent of cell hypertrophy. RA treatment also led to increased alkaline phosphatase activity occurring as early as 24 h after the start of treatment. The data in this study indicate that RA may have a role in endochondral ossification as an inducer/promoter of maturation-associated traits during chondrocyte maturation.

CELL HYPERTROPHY AND TYPE X COLLAGEN SYNTHESIS IN CULTURED ARTICULAR CHONDROCYTES

Articular cartilage is a permanent tissue whose cells do not normally take part in the endochondral ossification process. To determine whether articular chondrocytes possess the potential to express traits associated with this process such as cell hypertrophy and type X collagen, chondrocytes were isolated from adult chicken tibial articular cartilage and maintained in long-term suspension cultures. As a positive control in these experiments, we used parallel cultures of chondrocytes from the caudal portion of chick embryo sternum. Both articular and sternal chondrocytes readily proliferated and progressively increased in size with time in culture. Many had undergone hypertrophy by 4-5 weeks. Analysis of medium-released collagenous proteins revealed that both articular and sternal chondrocytes initiated type X collagen synthesis between 3 and 4 weeks of culture; synthesis of this macromolecule increased with further growth. Immunofluorescence analysis of 5-week-old cultures showed that about 15% of articular chondrocytes and 30% of sternal chondrocytes produced type X collagen; strikingly, there appeared to be no obvious relationship between type X collagen production and cell size. The results of this study show that articular chondrocytes from adult chicken tibia possess the ability to express traits associated with endochondral ossification when exposed to a permissive environment. They suggest also that the process of cell hypertrophy and initiation of type X collagen synthesis are independently regulated both in articular and sternal chondrocytes.

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595–605, 2009.

Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes

BackgroundDuring endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes.ResultsUsing a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition.ConclusionOur results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate.

RAPID INDUCTION OF TYPE X COLLAGEN GENE EXPRESSION IN CULTURED CHICK VERTEBRAL CHONDROCYTES

During endochondral ossification, small rapidly proliferating chondrocytes mature into flattened disc-shaped cells and then into large round hypertrophic cells. These morphological changes are accompanied by a decrease in the rate of cell proliferation. Type X collagen synthesis is initiated during chondrocyte maturation and reaches very high levels in the hypertrophic cells. We have analyzed type X collagen gene expression in chick embryo vertebral chondrocytes that were allowed to mature in monolayer culture and were then switched to suspension culture. The resuspended chondrocytes changed in shape from flat to round and decreased the proliferation rate as they do in vivo. These events were accompanied by a rapid, dramatic increase in type X collagen gene expression at the levels of transcription, steady-state mRNA and protein synthesis, as well as an increase in the number of cells producing type X collagen. The amount of type X collagen gene expression in resuspended chondrocytes was comparable to that in mineralizing cartilage in vivo. These results indicate that events accompanying the switch from monolayer to suspension culture (for example, the change from a flat to a round shape and/or the decrease in proliferation rate) may play a role in the induction of type X collagen gene expression during chondrocyte maturation. Thus we have developed an in vitro system that appears to mimic the events occurring during in vivo chondrocyte maturation. This in vitro model may provide an ideal system for further examination of the parameters regulating chondrocyte maturation and type X collagen gene expression.

Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response element

Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half‐sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half‐sites function as two overlapping RAREs that share the middle half‐site. Ablation of the middle half‐site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half‐sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production. J. Cell. Biochem.

Attachment of human marrow stromal cells to titanium surfaces.

The attachment of human bone marrow stromal cells to titanium alloy (Ti6Al4V) surfaces was investigated. Titanium disks were polished and modified by surface roughening and by passivation in nitric add. Cell attachment to titanium surfaces and tissue culture plastic (TCP) was determined by tetrazolium bromide (MTT) assay at 2, 6, 24, and 48 hours after seeding. Cell proliferation was determined by thymidine incorporation. Attachment on titanium surfaces was 75.6% to 94.9% of attachment on TCP control. The difference between cell attachment on the TCP compared with smooth or rough titanium was statistically significant (P < .05). However, no statistically significant difference was found between attachment to TCP and passivated titanium. Cell proliferation on titanium surfaces after 24 hours was approximately 70% of proliferation on TCP. There was a statistically significant difference (P < .05) between proliferation on tissue culture and smooth and passivated titanium but not on rough titanium. These results indicate that titanium provides a surface that is conducive to cell attachment and that passivating titanium improves cell attachment, approaching levels seen with TCP, a surface specifically developed to enhance cell attachment. Increasing surface roughness results in improved cell proliferation on titanium.

Retinoids and Indian Hedgehog Orchestrate Long Bone Development

Long bone formation is a complex process that has been studied for decades (1–3). It initiates with the emergence, at specific times and sites, of mesenchymal cell condensations that are patterned by the concerted action of the zone of polarizing activity (ZPA), apical ectodermal ridge, and dorsal ectoderm. The condensed cells differentiate into chondrocytes that produce characteristic cartilage matrix components and give rise to readily identifiable cartilaginous elements. The chondrocytes within each element start a process of maturation, which includes a proliferative, prehypertrophic, hypertrophic, and post-hypertrophic phase, and become organized in growth plates. At the same time, diarthrodial synovial joints develop at each epiphyseal end. Once formed, hypertrophic cartilage is invaded by bone, marrow, and vascular progenitor cells from adjacent perichondrial tissues, is eroded, and is finally replaced by endochondral bone and marrow. In addition, perichondrial cells give rise to an intramembranous bone collar surrounding the elements, which is critical to determine diameter and shape of the shaft (1).Maturation, hypertrophy, blood vessel invasion, and ossification first occur in the diaphyseal region and then spread toward the opposing epiphyses with increasing developmental time. Thus, long bone formation requires multiple and topographically restricted events within the cartilaginous elements as well as coordinated events in perichondrial tissues. It is not fully understood how all these processes are set and regulated, what signaling molecules mediate cartilage-perichondrium communication and interactions, and how events in cartilage are coordinated with those in perichondrium.