Project 1: Mechanosomes Essential for Regulating Chondrocyte Differentiation

Principal Investigator: Ying-jie (Angie) Guan, MD, PhD

Abstract

Mechanical stress plays an important role in cartilage homeostasis not only during endochondral bone formation in limb development but also during skeletal repair in adult life. Cellular perception and transduction of mechanical stress within cartilaginous tissues is an important modulator of chondrocyte function. However, the identity of the intracellular multi-protein complexes that transduce mechanical signals (mechanosomes) in chondrocytes is unclear. The mammalian target of rapamycin (mTOR), a highly conserved protein kinase, is a central controller of growth by functioning as a sensor of cellular nutrient, energy levels and redox status. Our hypothesis is that the mTOR signaling complexes mTORC1 and mTORC2 also play a central role in mechanotransduction by being part of the mechanosomes in chondrocytes.

Using a 3D primary chondrocyte culture mechanical loading system, we found that mechanical loading activates both mTORC1 and mTORC2 signaling, as evidenced by increased phosphorylation of their downstream targets respectively. In addition, inhibition of the mTORC1 pathway via rapamycin treatment blocks mechanical induction of Indian hedgehog (Ihh), a central mediator of mechanical regulation of chondrocyte proliferation and differentiation, while selective inhibition of PKC-a, a downstream target of mTORC2 blocked mechanical stimulation of the expression of chondrocyte hypertrophic marker collagen X. Furthermore, deficiency of SH2-containing protein tyrosine phosphatase 2 (Shp2) enhances mechanical stimulation of mTORC1.

Therefore, we hypothesize that activation of the mTOR signaling pathway through both mTORC1 and mTORC2 is required for mechanical stimulation of chondrocyte proliferation and differentiation and that mechanical activation of the mTOR signaling is negatively regulated by Shp2 in the mechanosomes in chondrocytes.

To test this hypothesis we will perform the following aims:

• Specific Aim 1: Define whether activation of the mTORC1 cascade by cyclic loading is required for mechanical regulation of chondrocyte proliferation and differentiation through Ihh.
• Specific Aim 2: Determine whether mechanical activation of the mTORC2 cascade affects chondrocyte hypertrophy by regulating cytoskeleton assembly through PKC-a. Specific Aim 3: Determine whether Shp2 negatively regulates mechanical activation of the mTORC1 and mTORC2 signaling.

Relevance

Understanding in detail the important intracellular pathways activated by mechanical loading during chondrocyte differentiation, including the role of mTOR in mechanotransduction, will provide insight into molecular mechanisms underlying mechanical regulation of cartilage homeostasis, thereby benefiting cartilage tissue engineering and regenerative medicine.