Guang Zhou

Dimorphic effects of Notch signaling in bone homeostasis

Notch signaling is a key mechanism in the control of embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and, specifically, in bone homeostasis, remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis owing to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by upregulating the genes encoding cyclin D, cyclin E and Sp7 (osterix). The intracellular domain of Notch1 also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all Notch signaling in osteoblasts, generated by deletion of the genes encoding presenilin-1 and presenilin-2 in bone, is associated with late-onset, age-related osteoporosis, which in turn results from increased osteoblast-dependent osteoclastic activity due to decreased osteoprotegerin mRNA expression in these cells. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.

Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo.

The α1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte–specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive β-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2 +/− background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

Dominance of SOX9 function over RUNX2 during skeletogenesis

Mesenchymal stem cell-derived osteochondroprogenitors express two master transcription factors, SOX9 and RUNX2, during condensation of the skeletal anlagen. They are essential for chondrogenesis and osteogenesis, respectively, and their haploinsufficiency causes human skeletal dysplasias. We show that SOX9 directly interacts with RUNX2 and represses its activity via their evolutionarily conserved high-mobility-group and runt domains. Ectopic expression of full-length SOX9 or its RUNX2-interacting domain in mouse osteoblasts results in an osteodysplasia characterized by severe osteopenia and down-regulation of osteoblast differentiation markers. Thus, SOX9 can inhibit RUNX2 function in vivo even in established osteoblastic lineage. Finally, we demonstrate that this dominant inhibitory function of SOX9 is physiologically relevant in human campomelic dysplasia. In campomelic dysplasia, haploinsufficiency of SOX9 results in up-regulation of the RUNX2 transcriptional target COL10A1 as well as all three members of RUNX gene family. In summary, SOX9 is dominant over RUNX2 function in mesenchymal precursors that are destined for a chondrogenic lineage during endochondral ossification.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six α-chains (1–6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the α3(IV) and α4(IV) chains, together with the α5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed α1(IV) and α2(IV) collagen to the α3(IV), α4(IV) and α5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b−/− mice, expression of both α(3)IV and α(4)IV collagen is strongly diminished in GBM, whereas that of α1, α2 and α5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of α3(IV) and α4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

Dysregulation of chondrogenesis in human cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia caused by heterozygosity of mutations in human RUNX2. The disorder is characterized by delayed closure of the fontanel and hypoplastic clavicles that result from defective intramembranous ossification. However, additional features, such as short stature and cone epiphyses, also suggest an underlying defect in endochondral ossification. Here, we report observations of growth-plate abnormalities in a patient with a novel RUNX2 gene mutation, a single C insertion (1228insC), which is predicted to lead to a premature termination codon and thus to haploinsufficiency of RUNX2 and the CCD phenotype. Histological analysis of the rib and long-bone cartilages showed a markedly diminished zone of hypertrophy. Quantitative real-time reverse transcription-polymerase chain reaction analysis of limb cartilage RNA showed a 5-10-fold decrease in the hypertrophic chondrocyte molecular markers VEGF, MMP13, and COL10A1. Together, these data show that humans with CCD have altered endochondral ossification due to altered RUNX2 regulation of hypertrophic chondrocyte-specific genes during chondrocyte maturation.

ERK1 and ERK2 regulate chondrocyte terminal differentiation during endochondral bone formation

Chondrocytes in the epiphyseal cartilage undergo terminal differentiation prior to their removal through apoptosis. To examine the role of ERK1 and ERK2 in chondrocyte terminal differentiation, we generated Osterix (Osx)‐Cre; ERK1–/–; ERK2flox/flox mice (conditional knockout Osx [cKOosx]), in which ERK1 and ERK2 were deleted in hypertrophic chondrocytes. These cKOosx mice were grossly normal in size at birth, but by 3 weeks of age exhibited shorter long bones. Histological analysis in these mice revealed that the zone of hypertrophic chondrocytes in the growth plate was markedly expanded. In situ hybridization and quantitative real‐time PCR analyses demonstrated that Matrix metalloproteinase‐13 (Mmp13) and Osteopontin expression was significantly decreased, indicating impaired chondrocyte terminal differentiation. Moreover, Egr1 and Egr2, transcription factors whose expression is restricted to the last layers of hypertrophic chondrocytes in wild‐type mice, were also strongly downregulated in these cKOosx mice. In transient transfection experiments in the RCS rat chondrosarcoma cell line, the expression of Egr1, Egr2, or a constitutively active mutant of MEK1 increased the activity of an Osteopontin promoter, whereas the MEK1‐induced activation of the Osteopontin promoter was inhibited by the coexpression of Nab2, an Egr1 and Egr2 co‐repressor. These results suggest that MEK1‐ERK signaling activates the Osteopontin promoter in part through Egr1 and Egr2. Finally, our histological analysis of cKOosx mice demonstrated enchondroma‐like lesions in the bone marrow that are reminiscent of human metachondromatosis, a skeletal disorder caused by mutations in PTPN11. Our observations suggest that the development of enchondromas in metachondromatosis may be caused by reduced extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK MAPK) signaling.

The long and the short of it: developmental genetics of the skeletal dysplasias

The skeletal dysplasias are a large heterogeneous group of genetic conditions characterized by abnormal shape, growth, or integrity of bones. Often, there may be prominent features associated with other organ systems as part of a more encompassing skeletal malformation syndrome. Tremendous advances have been made in the clinical and molecular delineation of these conditions over the past 20–30 years. We have progressed from initial broad clinical classifications of these conditions in the first two‐thirds of this century, to extensive delineation based on radiographic features in the 1970s and 1980s, to the present reconsideration and grouping of these conditions according to their molecular pathogenesis. This has in part been spurred on by advances in the understanding of the developmental pathways which govern skeletal development, as well as by the human genome sequencing effort, which has provided a plethora of positional candidate genes for many of these conditions. The pathogenetic correlations derived from such studies are often based on parallels between the human phenotype and mouse models of the human condition, and have sometimes revealed novel developmental functions.

Localization of the cis-enhancer element for mouse type X collagen expression in hypertrophic chondrocytes in vivo.

The type X collagen gene (Col10a1) is a specific molecular marker of hypertrophic chondrocytes during endochondral bone formation. Mutations in human COL10A1 and altered chondrocyte hypertrophy have been associated with multiple skeletal disorders. However, until recently, the cis‐enhancer element that specifies Col10a1 expression in hypertrophic chondrocytes in vivo has remained unidentified. Previously, we and others have shown that the Col10a1 distal promoter (−4.4 to −3.8 kb) may harbor a critical enhancer that mediates its tissue specificity in transgenic mice studies. Here, we report further localization of the cis‐enhancer element within this Col10a1 distal promoter by using a similar transgenic mouse approach. We identify a 150‐bp Col10a1 promoter element (−4296 to −4147 bp) that is sufficient to direct its tissue‐specific expression in vivo. In silico analysis identified several putative transcription factor binding sites including two potential activator protein‐1 (AP‐1) sites within its 5′‐ and 3′‐ends (−4276 to −4243 and −4166 to −4152 bp), respectively. Interestingly, transgenic mice using a reporter construct deleted for these two AP‐1 elements still showed tissue‐specific reporter activity. EMSAs using oligonucleotide probes derived from this region and MCT cell nuclear extracts identified DNA/protein complexes that were enriched from cells stimulated to hypertrophy. Moreover, these elements mediated increased reporter activity on transfection into MCT cells. These data define a 90‐bp cis‐enhancer required for tissue‐specific Col10a1 expression in vivo and putative DNA/protein complexes that contribute to the regulation of chondrocyte hypertrophy. This work will enable us to identify candidate transcription factors essential both for skeletal development and for the pathogenesis of skeletal disorders.

Mutation in lmx1b Causes Skeletal and Renal Dysplasia in Mice and Nail Patella Syndrome in Humans |[bull]| 710

Mutation in lmx1b Causes Skeletal and Renal Dysplasia in Mice and Nail Patella Syndrome in Humans • 710