Structure-based mutation analysis shows the importance of LRP5 β-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling

Bhat, Bheem M.; Allen, Kristina M.; Liu, Wei; Graham, James; Morales, Art; Anisowicz, Anthony; Lam, Hieu; McCauley, Catherine; Coleburn, Valerie E.; Cain, Michael; Fortier, Eric; Bhat, Ramesh A.; Bex, Frederick J.; Yaworsky, Paul J.

doi:10.1016/j.gene.2006.12.014

Cited by 44 publications

(37 citation statements)

References 34 publications

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“…TCF-luc; Fig. 2B; Bhat et al 2007). In U2OS osteosarcoma cells, over-expression of ERRaCPGC-1a together activated the reporter.…”

Section: Resultsmentioning

confidence: 98%

“…U2OS and C3H10T1/2 cells were purchased from ATCC (Manassas, VA, USA). U2OS cells were cultured as described (Bhat et al 2007). C3H10T1/2 cells were cultured in DMEM with high glucose and Glutamax (Invitrogen) with 10% FBS.…”

Section: Tissue Culture and Other Materialsmentioning

confidence: 99%

“…CMV-GFP was purchased from Promega. 16!TCFluciferase has been previously described (Bhat et al 2007). Point mutants in pAdORI-FLAG-ERRa were generated using the Quick-Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions.…”

Section: Constructs and Adenovirusmentioning

confidence: 99%

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Estrogen-related receptor α regulates osteoblast differentiation via Wnt/β-catenin signaling

Auld¹,

Berasi²,

Liu³

et al. 2012

Journal of Molecular Endocrinology

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Based on its homology to the estrogen receptor and its roles in osteoblast and chondrocyte differentiation, the orphan nuclear receptor estrogen-related receptor a (ERRa (ESRRA)) is an intriguing therapeutic target for osteoporosis and other bone diseases. The objective of this study was to better characterize the molecular mechanisms by which ERRa modulates osteoblastogenesis. Experiments from multiple systems demonstrated that ERRa modulates Wnt signaling, a crucial pathway for proper regulation of bone development. This was validated using a Wnt-luciferase reporter, where ERRa showed co-activator-dependent (peroxisome proliferator-activated receptor gamma co-activator 1a, PGC-1a) stimulatory effects. Interestingly, knockdown of ERRa expression also enhanced WNT signaling. In combination, these data indicated that ERRa could serve to either activate or repress Wnt signaling depending on the presence or absence of its co-activator PGC-1a. The observed Wnt pathway modulation was cell intrinsic and did not alter b-catenin nuclear translocation but was dependent on DNA binding of ERRa. We also found that expression of active ERRa correlated with Wnt pathway effects on osteoblastic differentiation in two cell types, consistent with a role for ERRa in modulating the Wnt pathway. In conclusion, this work identifies ERRa, in conjunction with co-activators such as PGC-1a, as a new regulator of the Wnt-signaling pathway during osteoblast differentiation, through a cell-intrinsic mechanism not affecting b-catenin nuclear translocation.

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“…TCF-luc; Fig. 2B; Bhat et al 2007). In U2OS osteosarcoma cells, over-expression of ERRaCPGC-1a together activated the reporter.…”

Section: Resultsmentioning

confidence: 98%

Section: Tissue Culture and Other Materialsmentioning

confidence: 99%

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Estrogen-related receptor α regulates osteoblast differentiation via Wnt/β-catenin signaling

Auld¹,

Berasi²,

Liu³

et al. 2012

Journal of Molecular Endocrinology

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“…These findings also point to the importance of the first WYTD-EGF ␤-propeller domain in regulating sensitivity of LRP5/6 to DKK1. Interestingly, all HBM mutations in LRP5 identified to date as well as the critical residue (Ser 243) required for mAb135 binding in LRP6, map to loops located on the upper face of the first WYTD-EGF ␤-propeller domain (Bhat et al, 2007). .…”

Section: Discussionmentioning

confidence: 99%

The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1

Binnerts

Tomašević

Bright

et al. 2009

MBoC

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The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.

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“…Structural analysis of the LRP5 protein revealed that all amino acids involved in any of the HBM are clustered at an open binding pocket near the surface of the first b-propeller of LRP5 (119). The mutations do not have any effect on the functioning of the protein but rather disrupt the ligand binding of the extracellular inhibitors DKK1 and sclerostin (120,121,122), in which a short binding motif was found by structure analysis (123).…”

Section: Identification Of the Role Of The Lrp5 Gene In Bonementioning

confidence: 98%

Genetics in Endocrinology: Autosomal dominant osteopetrosis revisited: lessons from recent studies

Bollerslev

Henriksen

Nielsen

et al. 2013

European Journal of Endocrinology

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Systematic studies of autosomal dominant osteopetrosis (ADO) were followed by the identification of underlying mutations giving unique possibilities to perform translational studies. What was previously designated ADO1 turned out to be a high bone mass phenotype caused by a missense mutation in the first propeller of LRP5, a region of importance for binding inhibitory proteins. Thereby, ADO1 cannot be regarded as a classical form of osteopetrosis but must now be considered a disease of LRP5 activation. ADO (Albers-Schönberg disease, or previously ADO2) is characterized by increased number of osteoclasts and a defect in the chloride transport system (ClC-7) of importance for acidification of the resorption lacuna (a form of Chloride Channel 7 Deficiency Osteopetrosis). Ex vivo studies of osteoclasts from ADO have shown that cells do form normally but have reduced resorption capacity and an expanded life span. Bone formation seems normal despite decreased osteoclast function. Uncoupling of formation from resorption makes ADO of interest for new strategies for treatment of osteoporosis. Recent studies have integrated bone metabolism in whole-body energy homeostasis. Patients with ADO may have decreased insulin levels indicating importance beyond bone metabolism. There seems to be a paradigm shift in the treatment of osteoporosis. Targeting ClC-7 might introduce a new principle of dual action. Drugs affecting ClC-7 could be antiresorptive, still allowing ongoing bone formation. Inversely, drugs affecting the inhibitory site of LRP5 might stimulate bone formation and inhibit resorption. Thereby, these studies have highlighted several intriguing treatment possibilities, employing novel modes of action, which could provide benefits to the treatment of osteoporosis.

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Structure-based mutation analysis shows the importance of LRP5 β-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling

Cited by 44 publications

References 34 publications

Estrogen-related receptor α regulates osteoblast differentiation via Wnt/β-catenin signaling

Estrogen-related receptor α regulates osteoblast differentiation via Wnt/β-catenin signaling

The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1

Genetics in Endocrinology: Autosomal dominant osteopetrosis revisited: lessons from recent studies

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