Runx2 and bone morphogenic protein 2 regulate the expression of an alternative Lef1 transcript during osteoblast maturation.

Lymphoid Enhancer Binding Factor (Lef) 1 is a transcriptional effector of the Wnt/Lrp5/beta-catenin signaling cascade, which regulates osteoblast differentiation, bone density, and skeletal strength. In this study, we describe the expression and function of an alternative Lef1 isoform in osseous cells. Lef1DeltaN is a naturally occurring isoform driven by a promoter (p2) within the intron between exons 3 and 4 of Lef1. Lef1DeltaN is induced during late osteoblast differentiation. This is converse to the expression pattern of the full-length Lef1 protein, which as we previously showed, decreases during differentiation. Agonists of osteoblast maturation differentially affected Lef1DeltaN expression. BMP2 stimulated Lef1DeltaN expression, whereas Wnt3a blocked basal and BMP2-induced expression of Lef1DeltaN transcripts during osteoblast differentiation. We determined that the Lef1DeltaN p2 promoter is active in osteoblasts and Runx2 regulates its activity. Stable overexpression of Lef1DeltaN in differentiating osteoblasts induced the expression of osteoblast differentiation genes, osteocalcin and type 1 collagen. Taken together, our results suggest Lef1DeltaN is a crucial regulator of terminal differentiation in osseous cells.

Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation.

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, both of which are characterized by craniofacial abnormalities, nearsightedness and hearing deficiencies. Despite its link to human diseases, few studies have described factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the beta-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted suppression of Col11a1 with an antisense morpholino accelerated osteoblastic differentiation and mineralization in C2C12 cells, similar to what was observed in Lef1-suppressed MC3T3 cells. Moreover incubation with a purified Col11a1 N-terminal fragment, V1B, prevented alkaline phosphatase expression in MC3T3 and C2C12 cells. These results suggest that Lef1 is an activator of the Col11a1 promoter and that Col11a1 suppresses terminal osteoblast differentiation.

Cell cycle related modulations in Runx2 protein levels are independent of lymphocyte enhancer-binding factor 1 (Lef1) in proliferating osteoblasts.

Runt-related transcription factor Runx2 regulates osteogenic phenotype commitment and attenuates osteoblast growth. Runx2 levels are cell cycle regulated and maximal in the G1 phase of proliferating osteoblasts and during quiescence. The Wnt/Lrp5-Frizzled/beta-catenin/Lef-Tcf signaling cascade also controls progression along the osteogenic lineage with a net anabolic effect that promotes bone formation. However, Lef1 opposes the osteoblast maturation promoting activity of Runx2. Here we examined whether Lef1 controls Runx2 expression during the cell cycle or onset of quiescence in osteoblasts. We inhibited Lef1 expression using short hairpin (sh) RNA interference in stably transfected MC3T3-E1 cells. In asynchronously growing osteoblasts, expression of Lef1 shRNA diminishes Lef1 protein levels, but does not affect Runx2 levels. Cells arrested in different cell cycle stages using mimosine (late G1), hydroxyurea or aphidicolin (S phase) or nocodazole (mitosis) exhibit expected reductions in Runx2 protein levels despite reductions in Lef1. Serum deprived MC3T3-E1 cells normally upregulate Runx2 protein regardless of Lef1 deficiency, although loss of Lef1 reduces cyclin A and increases cyclin D1 expression upon serum withdrawal. Thus, Runx2 protein levels during the cell cycle and onset of quiescence are regulated independently of Lef1, one of the major transcriptional inducers of Wnt signaling in proliferating cells.

Lymphocyte enhancer-binding factor 1 (Lef1) inhibits terminal differentiation of osteoblasts.

Lef1 is a transcriptional regulator of the Wnt/beta-catenin signaling cascade. Wnts directly augment bone formation and osteoblast differentiation from mesenchymal stem cells by receptor-mediated pathways involving Lrp5 and Frizzled. We previously reported that Lef1 represses Runx2-dependent activation of the late osteoblast differentiation gene, osteocalcin. Lef1 is expressed in preosteoblasts but is undetectable in fully differentiated osteoblasts. To determine if downregulation of Lef1 is necessary for osteoblast maturation, we constitutively overexpressed Lef1 in MC3T3-E1 preosteoblasts. Lef1-overexpressing cells produced alkaline phosphatase (ALP) and osteocalcin later, and at lower levels than control cells. Moreover, the extracellular matrices of Lef1-overexpressing cell cultures never mineralized. To further examine the role of Lef1 in osteoblasts, we suppressed Lef1 expression in MC3T3-E1 cells by RNA interference. Transient expression of a Lef1 shRNA efficiently reduced murine Lef1 levels and transcriptional activity. Stable suppression of Lef1 in MC3T3 preosteoblasts did not affect proliferation or Runx2 levels; however, ALP production and matrix mineralization were accelerated by 3-4 days. Gene chip analyses identified 14 genes that are differentially regulated in Lef1-suppressed cells. These data outline a role for Lef1 in delaying osteoblast maturation and suggest that Lef1 controls the expression of multiple genes in osteoblasts.

Wnt signaling in osteoblasts and bone diseases.

Recent revelations that the canonical Wnt signaling pathway promotes postnatal bone accrual are major advances in our understanding of skeletal biology and bring tremendous promise for new therapeutic treatments for osteoporosis and other diseases of altered bone mass. Wnts are soluble glycoproteins that engage receptor complexes composed of Lrp5/6 and Frizzled proteins. A subgroup of Wnts induces a cascade of intracellular events that stabilize beta-catenin, facilitating its transport to nuclei where it binds Lef1/Tcf transcription factors and alters gene expression to promote osteoblast expansion and function. Natural extracellular Wnt antagonists, Dickkopfs and secreted frizzled-related proteins, impair osteoblast function and block bone formation. In several genetic disorders of altered skeletal mass, mutations in LRP5 create gain-of-function or loss-of-function receptors that are resistant to normal regulatory mechanisms and cause higher or lower bone density, respectively. In this review, we summarize the available molecular, cellular, and genetic data that demonstrate how Lrp5 and other components of the Wnt signaling pathway influence osteoblast proliferation, function, and survival. We also discuss regulatory mechanisms discovered in developmental and tumor models that may provide insights into novel therapies for bone diseases.

Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation.

The Runt domain transcription factor Runx2 (AML-3, and Cbfa1) is essential for osteoblast development, differentiation, and bone formation. Runx2 positively or negatively regulates osteoblast gene expression by interacting with a variety of transcription cofactor complexes. In this study, we identified a trichostatin A-sensitive autonomous repression domain in the amino terminus of Runx2. Using a candidate approach, we found that histone deacetylase (HDAC) 3 interacts with the amino terminus of Runx2. In transient transfection assays, HDAC3 repressed Runx2-mediated activation of the osteocalcin promoter. HDAC inhibitors and HDAC3-specific short hairpin RNAs reversed this repression. In vivo, Runx2 and HDAC3 associated with the osteocalcin promoter. These data indicate that HDAC3 regulates Runx2-mediated transcription of osteoblast genes. Suppression of HDAC3 in MC3T3 preosteoblasts by RNA interference accelerated the expression of Runx2 target genes, osteocalcin, osteopontin, and bone sialoprotein but did not significantly alter Runx2 levels. Matrix mineralization also occurred earlier in HDAC3-suppressed cells, but alkaline phosphatase expression was not affected. Thus, HDAC3 regulates osteoblast differentiation and bone formation. Although HDAC3 is likely to affect the activity of multiple proteins in osteoblasts, our data show that it actively regulates the transcriptional activity of the osteoblast master protein, Runx2.

Tumor promoter-induced MMP-13 gene expression in a model of initiated epidermis.

In mouse epidermis in vivo, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases gene expression of matrix metalloproteinase-13 (MMP-13), an enzyme implicated in carcinogenesis. Here we used a keratinocyte cell line (308) derived from initiated mouse skin to investigate TPA-induced MMP-13 gene expression. Use of a pharmacological inhibitor (U0126) demonstrated that extracellular signal regulated kinase (ERK) plays a major role in TPA-induced MMP-13 gene expression. The 5'-flanking sequences of the MMP-13 gene contain binding sites for activator protein-1 (AP-1) and Runx. Both transcription factor families can be modulated by ERK and have been implicated in MMP-13 gene expression. TPA stimulated ERK-dependent increases in c-Fos protein and the c-Fos content of AP-1 complexes. MMP-13 promoter studies indicated that TPA requires AP-1, but not Runx, to induce MMP-13 gene expression. These studies show that in mouse keratinocytes MMP-13 gene expression can be induced through a Runx-independent pathway that involves the ERK-dependent modulation of AP-1.

Lymphoid enhancer factor-1 and beta-catenin inhibit Runx2-dependent transcriptional activation of the osteocalcin promoter.

Functional control of the transcription factor Runx2 is crucial for normal bone formation. Runx2 is detectable throughout osteoblast development and maturation and temporally regulates several bone-specific genes. In this study, we identified a novel post-translational mechanism regulating Runx2-dependent activation of the osteocalcin promoter. A functional binding site for the high mobility group protein lymphoid enhancer-binding factor 1 (LEF1) was found adjacent to the proximal Runx2-binding site in the osteocalcin promoter. In transcription assays, LEF1 repressed Runx2-induced activation of the mouse osteocalcin 2 promoter in several osteoblast lineage cell lines. Mutations in the LEF1-binding site increased the basal activity of the osteocalcin promoter; however, the LEF1 recognition site in the osteocalcin promoter was surprisingly not required for LEF1 repression. A novel interaction between the DNA-binding domains of Runx2 and LEF1 was identified and found crucial for LEF1-mediated repression of Runx2. LEF1 is a nuclear effector of the Wnt/LRP5/beta-catenin signaling pathway, which is also essential for osteoblast proliferation and normal skeletal development. A constitutively active beta-catenin enhanced LEF1-dependent repression of Runx2. These data identify a novel mechanism of regulating Runx2 activity in osteoblasts and link Runx2 transcriptional activity to beta-catenin signaling.

Runx2 (Cbfa1, AML-3) interacts with histone deacetylase 6 and represses the p21(CIP1/WAF1) promoter.

Runx2 (Cbfa1, AML-3) is multifunctional transcription factor that is essential for osteoblast development. Runx2 binds specific DNA sequences and interacts with transcriptional coactivators and corepressors to either activate or repress transcription of tissue-specific genes. In this study, the p21(CIP/WAF1) promoter was identified as a repressible target of Runx2. A carboxy-terminal repression domain distinct from the well-characterized TLE/Groucho-binding domain contributed to Runx2-mediated p21 repression. This carboxy-terminal domain was sufficient to repress a heterologous GAL reporter. The repressive activity of this domain was sensitive to the histone deacetylase inhibitor trichostatin A but not to trapoxin B. HDAC6, which is insensitive to trapoxin B, specifically interacted with the carboxy terminus of Runx2. The HDAC6 interaction domain of Runx2 was mapped to a region overlapping the nuclear matrix-targeting signal. The Runx2 carboxy terminus was necessary for recruitment of HDAC6 from the cytoplasm to chromatin. HDAC6 also colocalized and coimmunoprecipitated with the nuclear matrix-associated protein Runx2 in osteoblasts. Finally, we show that HDAC6 is expressed in differentiating osteoblasts and that the Runx2 carboxy terminus is necessary for maximal repression of the p21 promoter in preosteoblasts. These data identify Runx2 as the first transcription factor to interact with HDAC6 and suggest that HDAC6 may bind to Runx2 in differentiating osteoblasts to regulate tissue-specific gene expression.