Sox9
Role of the Transcriptor Factor SOX9 in tracheal cartilage Development.
Knowledge of the molecular mechanisms that orchestrate this complex algorithm of cellular differentiation and spatial organization, and gives rise to the upper respiratory tract is essentially non-existent. To understand the molecular mechanisms of upper respiratory tract cartilage development, it is therefore necessary to make inferences from other organ systems. Recently, the transcription factor, Sox9, has surfaced as a gene that may play a key role in the chondrogenesis of skeletal structures. Specifically, the identification of a heterozygous mutation in the Sox9 gene in a rare human genetic disease called Campomelic syndrome (or campomelic dysostosis; MIM *114290) suggested a role for this gene in chondrocyte differentiation . Campomelic syndrome is an autosomal dominant skeletal malformation characterized by shortness and bowing of long bones, especially of the lower limbs. Additional findings include 11 pairs of ribs and a bell shaped thorax, hypoplastic scapulae, narrow iliac wings, non-mineralized thoracic pedicles, clubbed feet, Robin Sequence, and typical facial anomalies . Sex reversal occurs in 75% of patients with an XY karyotype. Interestingly, tracheobronchiomalacia, and hypoplastic lungs are often found in Campomelic syndrome, contributing to respiratory distress and frequent lethality in the neonatal period .
Sox9 is a member of the Sox (Sry-type HMG box) subfamily of proteins containing a DNA-binding high-mobility-group (HMG) domain. The HMG domain of Sox proteins has at least 50% amino acid sequence identity with testis-determining factor (Sry) and with each other. To date greater than 20 sox proteins have been identified in vertebrates. Sox proteins show highly restricted expression patterns and are involved in the regulation of such diverse developmental processes as germ layer formation, organ development and cell type specification . The Sox9 gene located on human chromosome 17 and mouse chromosome 11 is expressed prominently in chondrogenic precursor cells . In addition, Sox9 is expressed in the genital ridge and adult testis, ventricular CNS cells, notochord, otocysts, tubular heart structures, kidney, pancreas and vibrissae. Though evidence from patients with Campomelic Syndrome suggests that a heterozygous mutation in Sox9 results in congenital upper respiratory tract anomalies , the temporal-spatial expression of Sox9 in the upper respiratory tract has not been characterized.
In addition to the skeletal anomalies of Campomelic syndrome and the expression of Sox9 in chondrocytes, there are several lines of evidence demonstrating that this gene is important in chondrocyte differentiation. Sox9 bound and strongly activated the 48-bp chondrocyte-specific collagen II (Col2a1) enhancer element . Ectopic expression of Sox9 in transgenic animals resulted in ectopic expression of Col2A1 . Mutations that abolish Sox9 binding rendered these enhancers inactive in transgenic mice . In addition, Sox9 also bound to the promoter of other chondrocyte marker genes such as Collagen XI (Col11a2), and mutations in the Sox9 binding sites in these promoters abolished their chondrocyte-specific activity . These lines of evidence suggested that cartilage specific genes such as Col2A1 and Col11a2 are the direct targets of Sox9.
To determine the role of Sox9 in chondrocyte differentiation, Bi et al constructed a heterozygous Sox9 mutant mouse . The Sox9+/- mice died perinatally, showing evidence of hypoplasia and bending of many skeletal and cartilaginous structures. The hyoid bone, laryngeal cartilage and tracheal cartilage were among the hypoplastic entities noted in these mutant mice , though the abnormal morphology of these structures was not carefully analyzed. Interestingly, there were no obvious patterning defects, suggesting that Sox9 haploinsufficiency did not effect skeletal-pattern formation. By examining the development of cartilage in Sox9+/- mice, hypoplastic cartilage was found to be a result of hypoplastic mesenchymal condensations or cartilage primordia. Hypoplastic mesenchymal condensations were not a result of decreased proliferation rates as demonstrated by bromodeoxyuridine labeling. Therefore, Sox9 haploinsufficiency appeared to block recruitment of cells to mesenchymal condensations, one of the earliest steps in chondrocyte differentiation. This hypothesis is verified in Sox9 knockout chimeras, in which cells that do not contain the Sox9 gene are excluded from cartilage, but are present as juxtaposed mesenchyme that does not express chondrocyte specific markers . An explanation for the effect of Sox9 haploinsufficiency may lie in the fact that the dissociation constant (Kd) for Sox9 is in the nanomolar range, in contrast to the Kd of many other transcription factors that are in the picomolar range. Therefore it is possible that the absolute concentration of Sox9 is near the Kd value in wild-type animals, and thus a 50% reduction in Sox9 would affect transcription of at least some Sox9-dependent genes.
Two other members of the Sox family, L-Sox5 and Sox6, and Lc-Maf, a proto-oncoprotein have also been shown to bind to the Col2a1 enhancer element. The two Sox family members, L-Sox5 and Sox6, do not bear any identity to Sox9 except for in the HMG box. All three transcription factors are coexpressed with Sox9 in areas of precartilaginous mesenchymal condensations. In addition, L-Sox5, Sox6 and Lc-Maf cooperate with Sox9 to activate the Col2A1 enhancer element. The role of these transcription factors in chondrocyte differentiation has yet to be completely understood.
Since Sox9 appears to be essential for chondrocyte differentiation, it is not unreasonable to speculate that its expression might be a target of signaling molecules like fibroblast growth factors (FGFs) that are known to influence discrete steps in chondrogenesis. The role of FGFs in various processes of embryonic development, including the earliest stages of limb development and cartilage development has been established . The current 22 members of the FGF family can be grouped into subfamilies based on greater sequence similarity and can activate one of four high-affinity FGF receptor (FGFR) tyrosine kinases . FGFR1, FGFR2 and FGFR4, are expressed in chondrocyte primordia, and FGFR3 is expressed in the resting and proliferating zones of cartilages in the growth plate . Furthermore, genetic linkage analysis has implicated three members of the FGFR family (FGFR1 to 3) as the underlying cause of several skeletal dysplasias and autosomal dominant craniosynostosis syndromes . This evidence strongly suggests a role for FGFs in chondrogenesis, though the specific pathways of this influence are currently unknown.
A specific FGF, FGF18, has recently gained attention as potentially playing a key role in chondrogenesis. FGF18 is expressed in chondrocytes at different stages of development . Mice homozygous for a targeted disruption of FGF18 or that conditionally over-express FGF18 had an abnormal cartilage phenotype . In fact mice that over-expressed FGF18 displayed abnormal tracheal cartilage rings. Equally interesting, Murakami et al recently demonstrated that FGF18 could upregulate Sox9 expression via a mitogen-activated protein kinase pathway. Taken together, these studies suggest that FGF18 could affect cartilage development by regulating the temporal-spatial expression of Sox9. This hypothesis has not yet been substantiated.