Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice

Stefano Zanotti; Ernesto Canalis

doi:10.1074/jbc.M113.489435

Hairy and Enhancer of Split-related with YRPW Motif (HEY)2 Regulates Bone Remodeling in Mice*

From the ^‡Department of Research, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105-1299 and
the ^§University of Connecticut School of Medicine, Farmington, Connecticut 06030

↵1 To whom correspondence should be addressed: Dept. of Research, Saint Francis Hospital and Medical Center, 114 Woodland St., Hartford, CT 06105-1299. Tel.: 860-714-4068; Fax: 860-714-8053; E-mail: ecanalis{at}stfranciscare.org.

Background: Notch regulates bone mass and induces Hairy and Enhancer of Split-related with YRPW motif (HEY) in osteoblasts, but the skeletal function of individual HEY proteins is unclear.

Results: In male mice, Hey2 inactivation increases bone mass, whereas HEY2 overexpression enhances bone resorption, reducing bone mass.

Conclusion: HEY2 regulates skeletal remodeling in male mice, decreasing bone mass.

Significance: HEY2 mediates selected skeletal effects of Notch.

Abstract

Notch induces Hairy and Enhancer of Split-related with YRPW motif (Hey)1, Hey2, and HeyL expression in osteoblasts, but the contributions of these genes to the skeletal effects of Notch are not fully understood. HEY1 misexpression has limited skeletal impact, female HeyL null mice display increased bone mass, and Hey2 inactivation is developmentally lethal. To inactivate Hey2 in immature or mature osteoblasts, Hey2^loxP/loxP mice were crossed with transgenics expressing CRE under the control of the osterix (Osx-Cre) or osteocalcin (Oc-Cre) promoters to generate Osx-Cre^+/−;Hey2^Δ/Δ or Oc-Cre^+/−;Hey2^Δ/Δ mice. Trabecular bone volume increased in 3-month-old Osx-Cre^+/−;Hey2^Δ/Δ and Oc-Cre^+/−;Hey2^Δ/Δ male mice and in 1-month-old Oc-Cre^+/−;Hey2^Δ/Δ female mice, although 3-month-old Oc-Cre^+/−;Hey2^Δ/Δ females developed osteopenia. Alkaline phosphatase liver/bone/kidney (ALPL) expression and activity were suppressed in osteoblasts from Oc-Cre^+/−;Hey2^Δ/Δ mice of both sexes. To overexpress HEY2 in osteoblasts, transgenic mice where a 3.6-kb fragment of the rat collagen type-I α1 promoter directs HEY2 expression were created. Three-month-old Hey2 transgenic males exhibited decreased osteoblast activity and increased bone resorption and developed osteopenia at 6 months of age. Hey2 transgenic females exhibited reduced osteoblast number and function, but no changes in bone resorption. HEY2 overexpression in osteoblasts from mice of both sexes inhibited ALPL expression and activity and suppressed osteocalcin transcripts in cells from male mice only. HEY2 overexpression in osteoblasts from male mice enhanced bone resorption by co-cultured splenocytes and induced interleukin-6, a molecule that promotes osteoclastogenesis. In conclusion, HEY2 decreases skeletal mass and regulates bone remodeling in male mice.

Footnotes

↵* This work was supported, in whole or in part, by National Institutes of Health Grant DK045227 from the NIDDK (to E. C.) and Research Fellowship Award 5371 from the Arthritis Foundation (to S. Z.).