scholarly journals Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA binding of Runx2

Development ◽  
2009 ◽  
Vol 136 (4) ◽  
pp. 615-625 ◽  
Author(s):  
N. Funato ◽  
S. L. Chapman ◽  
M. D. McKee ◽  
H. Funato ◽  
J. A. Morris ◽  
...  
Keyword(s):  
Dna Binding ◽  
Osteoblast Differentiation ◽  
Branchial Arch

Physical and genetic interactions between Alx4 and Cart1

Development ◽  
1999 ◽  
Vol 126 (2) ◽  
pp. 359-369 ◽  
Author(s):  
S. Qu ◽  
S.C. Tucker ◽  
Q. Zhao ◽  
B. deCrombrugghe ◽  
R. Wisdom
Keyword(s):  
Dna Binding ◽  
Double Mutant ◽  
Biochemical Characterization ◽  
Gene Dosage ◽  
Genetic Relationships ◽  
Branchial Arch ◽  
Genetic Interactions ◽  
Limb Bud ◽  
High Affinity

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.

The Transcription Repressor Gfi1 Directly Interacts With and Is Phosphorylated By Aurora A Kinase, Which Abrogates Myeloma-Induced Gfi1 Repression Of The Runx2 Promoter In Pre-Osteoblasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1844-1844
Author(s):  
Jixin Ding ◽  
Fengming Wang ◽  
ShunQian Jin ◽  
Judy Anderson ◽  
Deborah L. Galson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Disease ◽  
Stromal Cells ◽  
Protein Turnover ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Aurora A Kinase

Abstract Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

scholarly journals Inhibition of BMP2-Induced Bone Formation by the p65 Subunit of NF-κB via an Interaction With Smad4

2014 ◽  
Vol 28 (9) ◽  
pp. 1460-1470 ◽  
Author(s):  
Shizu Hirata-Tsuchiya ◽  
Hidefumi Fukushima ◽  
Takenobu Katagiri ◽  
Satoshi Ohte ◽  
Masashi Shin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Bone Diseases ◽  
Bmp Signaling ◽  
Wild Type ◽  
Smad Pathway ◽  
Wild Type Mefs

Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11–770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65−/− MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.

The Transcription Repressor Gfi1 Directly Interacts With and Is Phosphorylated By Aurora A Kinase, Which Abrogates Myeloma-Induced Gfi1 Repression Of The Runx2 Promoter In Pre-Osteoblasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1184-1184
Author(s):  
Jixin Ding ◽  
Fengming Wang ◽  
ShunQian Jin ◽  
Judy Anderson ◽  
Deborah L. Galson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Disease ◽  
Stromal Cells ◽  
Protein Turnover ◽  
Bone Marrow Stromal Cells ◽  
Osteoblast Differentiation ◽  
Marrow Stromal Cells ◽  
Aurora A Kinase

Abstract Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

scholarly journals Up-regulation of inhibitors of DNA binding/differentiation gene during alendronate-induced osteoblast differentiation

2011 ◽  
Vol 285 (5) ◽  
pp. 1331-1338 ◽  
Author(s):  
Ae Ra Kang ◽  
Young Rim Oh ◽  
Heung Yeol Kim ◽  
Min Jung Park ◽  
Bo Sun Joo ◽  
...  
Keyword(s):  
Dna Binding ◽  
Osteoblast Differentiation ◽  
Differentiation Gene
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