Hicham Drissi

Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene

The runt related transcription factor CBFA1 (AML3/PEBP2αA/RUNX2) regulates expression of several bone‐ and cartilage‐related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5′ regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone‐related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt −351 and −92 causes a striking 30‐ to 100‐fold combined decrease in promoter activity. Additionally, 5′ UTR sequences repress reporter gene transcription 2‐ to 3‐fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5′ region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5′ UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation. J. Cell. Physiol. 184:341–350, 2000.

Phenotype discovery by gene expression profiling: Mapping of biological processes linked to BMP-2-mediated osteoblast differentiation

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP‐2, a member of the TGFβ superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP‐mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP‐2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP‐2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP‐2 and expression was modulated from 3‐ to 14‐fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up‐ or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP‐2 immediately responsive genes (1–4 h), which exhibited either transient or sustained expression, reflect activation and repression of non‐osseous BMP‐2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2–8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic‐related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis identified a multistage molecular and cellular cascade that supports BMP‐2‐mediated osteoblast differentiation. J. Cell. Biochem. 89: 401–426, 2003.

Wnt induction of chondrocyte hypertrophy through the Runx2 transcription factor

We investigated the molecular mechanisms underlying canonical Wnt‐mediated regulation of chondrocyte hypertrophy using chick upper sternal chondrocytes. Replication competent avian sarcoma (RCAS) viral over‐expression of Wnt8c and Wnt9a, upregulated type X collagen (col10a1) and Runx2 mRNA expression thereby inducing chondrocyte hypertrophy. Wnt8c and Wnt9a strongly inhibited mRNA levels of Sox9 and type II collagen (col2a1). Wnt8c further enhanced canonical bone morphogenetic proteins (BMP‐2)‐induced expression of Runx2 and col10a1 while Wnt8c and Wnt9a inhibited TGF‐β‐induced expression of Sox9 and col2a1. Over‐expression of β‐catenin mimics the effect of Wnt8c and Wnt9a by upregulating Runx2, col10a1, and alkaline phosphatase (AP) mRNA levels while it inhibits col2a1 transcription. Western blot analysis shows that Wnt8c and β‐catenin also induces Runx2 protein levels in chondrocytes. Thus, our results indicate that activation of the canonical β‐catenin Wnt signaling pathway induces chondrocyte hypertrophy and maturation. We further investigated the effects of β‐catenin‐TCF/Lef on Runx2 promoter. Co‐transfection of lymphoid enhancer factor (Lef1) and β‐catenin in chicken upper sternal chondrocytes together with deletion constructs of the Runx2 promoter shows that the proximal region spanning the first 128 base pairs of this promoter is responsible for the Wnt‐mediated induction of Runx2. Mutation of the TCF/Lef binding site in the −128 fragment of the Runx2 promoter resulted in loss of its responsiveness to β‐catenin. Additionally, gel‐shift assay analyses determined the DNA/protein interaction of the TCF/Lef binding sites on the Runx2 promoter. Finally, our site‐directed mutagenesis data demonstrated that the Runx2 site on type X collagen promoter is required for canonical Wnt induction of col10a1. Altogether we demonstrate that Wnt/β‐catenin signaling is regulated by TGF‐β and BMP‐2 in chick upper sternal chondrocytes, and mediates chondrocyte hypertrophy at least partly through activation of Runx2 which in turn may induce col10a1 expression. J. Cell. Physiol.

Reduced COX-2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX‐2), the inducible regulator of prostaglandin E2 (PGE2) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX‐2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX‐2 is expressed primarily in early cartilage precursors that co‐express col‐2. COX‐2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX‐2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX‐2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging.

Cyclin D1-Cdk4 Induce Runx2 Ubiquitination and Degradation *

Runx2 is a Runt domain transcription factor involved in the activation of genes encoding osteoblast and chondrocyte-specific proteins. Runx2 activity is regulated by transcriptional and post-transcriptional mechanisms. The functional significance of the post-translational modification of Runx2 has not been fully defined. We show that cyclin D1-Cdk4 induce Runx2 degradation in an ubiquitination-proteasome-dependent manner. Mutagenesis of Runx2 serine-472, a consensus Cdk site, to alanine increases the half-life of Runx2 and causes loss of sensitivity to cyclin D1-induced Runx2 degradation. The targeted Runx2 degradation by cyclin D1 identifies a novel mechanism through which Runx2 activity is regulated coordinately with the cell cycle machinery in bone cells.

Murine and chicken chondrocytes regulate osteoclastogenesis by producing RANKL in response to BMP2.

Chondrocytes express RANKL, but their role in osteoclastogenesis is not clear. We report that hypertrophic chondrocytes induce osteoclast formation through RANKL production stimulated by BMP2 and Runx2/Smad1 and thus they may regulate resorption of calcified matrix by osteoclasts at growth plates.

Smad3-Deficient Chondrocytes Have Enhanced BMP Signaling and Accelerated Differentiation

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF‐β signaling factors, but BMP‐related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3−/− chondrocytes. BMP signaling is increased in TGF‐β deficiency and is required for accelerated chondrocyte maturation.

Rapid Isolation of Human Stem Cells (Connective Tissue Progenitor Cells) From the Proximal Humerus During Arthroscopic Rotator Cuff Surgery

Background: Bone-to-tendon healing in the shoulder can be unpredictable. Biologic augmentation, through the implementation of adult mesenchymal stem cells, may improve this healing process. Purpose: The purpose of this study was to (1) arthroscopically obtain bone marrow aspirates from the proximal humerus during rotator cuff repair, (2) purify and concentrate the connective tissue progenitor cells (CTPs) in the operating room efficiently, and (3)confirm these are stem cells through their ability to differentiate into bone cells. We hypothesize that CTPs can be quickly and efficiently isolated from bone marrow during arthroscopic surgery and that these cells are capable of osteogenesis. Study Design: Cohort study; Level of evidence, 3; and Descriptive laboratory study. Methods: Bone marrow aspirates were harvested through the anchor tunnel of the humeral head during arthroscopic rotator cuff repair in 23 patients. Twenty-three matched controls were selected from a clinical registry to evaluate for increased incidence of complication. Connective tissue progenitor cells were isolated using 2 accepted methods and compared with a novel, rapid method designed for use in the operating room. Osteogenic potential was assessed by cytochemical and molecular analysis. Results: Reverse transcription polymerase chain reaction analysis and cellular staining confirmed the osteogenic potential of these CTPs. There was no statistical significant difference in the Single Assessment Numeric Evaluation score (aspirate, 86.3 ± 10.5; control, 83.6 ± 15.1; P = .54), range of motion measures (postoperative external rotation: aspirate, 65.0° ± 20.4°; control, 62.5° ± 17.1°; P = .67; postoperative forward elevation: aspirate, 163.0° ± 30.6°; control, 145.7° ± 41.4°; P = .12), or postoperative strength measures between groups (median, 5; range, 4-5 in the aspirate group compared with median, 5; range, 4-5 in the control group; P > .05). Conclusion: Connective tissue progenitor cells can be safely and efficiently aspirated from the proximal humerus using the anchor tunnel created during arthroscopic rotator cuff surgery. These cells may play an important role in cell-based therapies involving rotator cuff repair. Clinical Relevance: We have established a reliable, reproducible protocol for isolating CTPs in the operating room. These cells may have the potential to enhance the healing process after rotator cuff repair.

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real‐time RT‐PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis.

Osterix/Sp7 regulates mesenchymal stem cell mediated endochondral ossification

We investigated the expression and regulation of the zinc finger protein Osterix (Osx) during endochondral ossification in mice. In studies to determine the temporal and spatial regulation of Osx mRNA and protein during embryogenesis we found it to be present throughout development, but its expression is restricted to the immature chondro/osteoprogenitor cells and mature osteoblasts, excluding hypertrophic chondrocytes. Using a fracture model, we show a consistent pattern of Osx protein expression in mesenchymal progenitor cells in the periosteum and immature chondrocytes and osteoblasts embedded in the fracture callus. In contrast, hypertrophic chondrocytes, vessels and fibrous tissue were devoid of Osx expression. Additionally, using RNA isolated from fracture callus throughout the healing process, we observe that Osx transcripts parallel that of Runx2 and differentially overlap both cartilage and bone phenotypic markers. Furthermore, using limb bud‐derived MLB13MYC Clone 17 cells, we show that PTHrP inhibited chondrocyte maturation while it enhanced mRNA levels of Osx in these chondro/osteoprogenitor cells. Gain and loss of function of Osx function experiments with these cells demonstrated that Osx serves as an inhibitor of chondrogenesis and chondrocyte maturation, while it promotes osteoblast maturation. Together, our findings provide the first demonstration of the molecular mechanisms underlying Osx inhibition of chondrocyte differentiation, and further suggest a role for this transcription factor in mediating endochondral ossification during bone repair. J. Cell. Physiol. 214:173–182, 2008.