Elucidation of the functions of Ywhag and Ywhae in bone development using their knockout mice

2017 Fiscal Year Research-status Report

• Project/Area Number: 17K17094
• Research Institution: Nagasaki University
• Principal Investigator: 姜 晴 長崎大学, 医歯薬学総合研究科(歯学系), 助教 (00790007)
• Project Period (FY): 2017-04-01 – 2019-03-31
• Keywords: 骨代謝学

Outline of Annual Research Achievements

The involvement of 14-3-3s in bone development is poorly understood, and only a few reports have been published. To investigate the functions of Ywhag and Ywhae in bone development in detail, I established two Ywhag-/- lines and two Ywhae-/- lines by injecting each gRNA. And we used these loss of function mouse models to clarify the physiological functions of Ywhag and Ywhae in bone development.
Wild-type and Ywhag-/-, Ywhae-/- samples were collected at E15.5 and E18.5. We stained whole skeletons with alcian blue and alizarin red to identify cartilage and calcified tissues, respectively. Ywhag-/- showed similar level compared with wild-type, while size of Ywhae-/- skeletons was smaller than wild-type. Due to embryonic lethality of Ywhae-/- mice, wild-type and Ywhag-/-, Ywhae+/- samples were obtained from the mice at 4 weeks and 8 weeks of age. Body weight and bone volume of Ywhag-/- mice and Ywhae+/- mice were lower than that of wild-type mice, especially at 4-6 weeks of age.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Because of embryonic lethality of Ywhae-/- mice, we can not analyze Ywhae-/- mice at adult stage, so we compared wild-type and Ywhae+/- mice by microCT at 8 weeks of age. We will aslo identify the reason of dwarfism in Ywhae+/- mice.

Strategy for Future Research Activity

The general functions of 14-3-3s were reported, but the functions on bone development are still unknown. This is the first study of 14-3-3s in bone development using KO mouse models. This study will greatly contribute to the elucidation of the molecular mechanisms for bone development.
We will compare Runx2 343-bp enhancer activities in primary osteoblasts from wild-type, Ywhag-/-, Ywhae-/- primary osteoblasts by reporter assay to identify the target molecules of the 14-3-3s in the activation of the Runx2 enhancer by introducing the transcription factors and cofactors, which had activated the enhancer.
As Runx2 is an important transcription factor for bone formation and homeostasis and is potentially useful for osteoporosis therapy, elucidation of the molecular mechanisms, by which 14-3-3s enhance the osteoblast-specific enhancer, will hopefully provide novel insights into the therapy of osteoporosis.

Causes of Carryover

理由 We already have data of these KO mice condition basically, we need to research details in bone development, to examine the functions of Ywhag and Ywhae in osteoblast, osteocyte and osteoclast.

使用計画We will use knockout mice as model to identify the function of Ywhag and Ywhae, we need ELISA kit to examine TRAP5b and Osteocalcin level in serum of these KO mice, we will isolate RNA and examine mRNA level of osteoblast and osteoclast marker genes.