Mice Deficient in AKAP13 Are Osteoporotic and Have Impaired Osteogenesis

Presentation Number: SAT 339
Date of Presentation: April 2nd, 2016

Hisashi Koide*1, Kenn Holmbeck2, Julian Lui3, Paul H Driggers4, Akitoshi Nakayama1, Tifferny Chu4, Ichiro Tatsuno5, Tomoaki Tanaka1, Koutaro Yokote1 and James H. Segars4
1Chiba University Graduate School of Medicine, Chiba, Japan, 2NIH, 3NIH, Bethesda, MD, 4Johns Hopkins School of Medicine, 5Toho University Sakura Medical Center, Kashiwa-City, Japan

Abstract

The Protein Kinase A Anchoring Protein 13 (Akap13) is a guanine nucleotide-exchange factor (GEF) for Rho-type small GTP-binding, and functions as a cytoplasmic integrator or docking platform for multiple signaling cascades including those of the protein kinase A and nuclear hormone receptors, such as estrogen and glucocorticoid receptors. All these signaling cascades are important for bone metabolism, while Rho-type small G proteins are essential for mediating mechanical stress, an important stimulator of bone formation. Thus, we examined involvement of Akap13 in the regulation of bone development by employing Akap13 haploinsufficient mice and in vitro analyses. Akap13 was expressed in bone, and mice haploinsufficient for Akap13 (Akap13t/–) displayed reduced bone mineral density, reduced bone volume/total volume, and trabecular number, and increased trabecular spacing;resembling the changes observed in osteoporotic bone. Consistent with the osteoporotic phenotype, Colony forming unit-fibroblast numbers were diminished in Akap13t/– mice, as were osteoblast numbers in periosteum and extracellular matrix production when compared to control littermates. Transcripts of Runx2, an essential transcription factor for the osteogenic lineage, and alkaline phosphatase (Alp), an indicator of osteogenic commitment, were both reduced in femora of Akap13t/– mice. Knockdown of Akap13 reduced levels of Runx2 and Alp transcripts in immortalized bone marrow stem cells. These findings suggest that Akap13 haploinsufficient mice have a deficiency in early osteogenesis with a corresponding reduction in osteoblast number, but no impairment of mature osteoblast activity. Notably, Akap13 has a unique structure of functional domains, such as GEF, PKA signaling and nuclear hormone receptor interacting domain (NRID). It is able to receive multiple intracellular signaling pathways. To further investigate how Akap13 functions to regulate osteogenesis, we generated the deletion mutant (mt) of Akap13 and then examined its effect on Alp expression. Transfection of human and mouse Akap13 wild type (wt) increased Alp mRNA in BMSCs 99.8% and 82.7% compared to control respectively, while induction of mt structures lucking PKA anchoring domain or GEF domain increased Alp mRNA 27.2% and 7.7% compared to control respectively but these effects were lower than that of wt. Transfection of NRID in carboxyl region does not affect Alp mRNA expression, suggesting that PKA and/or Rho signaling or additional stimulation of nuclear receptor hormone is necessary for functioning of NRID. Our results suggest that each signal is important for functioning of Akap13 on bone and Akap13 might integrate signal inputs from mechanical stress or extracellular conditions mediated by small G proteins and action of steroid hormones to Runx2-mediated bone formation.

 

Nothing to Disclose: HK, KH, JL, PHD, AN, TC, IT, TT, KY, JHS