Temporomandibular joints are constantly subjected to various types of mechanical loading, including compression, tension, and shear during mouth opening and closing movements (Kamiya et al., 2010). Mechanical loading affects the proliferation and differentiation of chondrocytes in vitro via mechanotransduction (Zhao et al., 2020). One of the most well-known mechanosensitive signaling pathways in chondrocytes is the hedgehog pathway (Bechtold et al., 2019). For example, the expression levels of Patched 1 (Ptch1) and Smoothened (Smo), the two key components of the hedgehog signaling axis, in chondrocytes were increased following 10% cyclic tensile strain for 1 h or 24 h at 0.33 Hz (Thompson et al., 2014). Malfunction of hedgehog signaling causes craniofacial skeletal abnormalities (Deng et al., 2019, Hammond et al., 2018). Especially, mice with a mesenchymal deletion of Smo, an essential molecule for the hedgehog signaling activity, show significantly shortened mandibles (Kitamura et al., 2020). Notably, the hedgehog protein initiates signal transduction by binding to the receptor Ptch1, which will release Smo out of primary cilia (Mukhopadhyay and Rohatgi, 2014). Smo mediates downstream signal transduction and activates the Glioma-associated oncogene homolog (Gli) protein at the ciliary tip (Liu 2019). Subsequently, the activated Gli protein is transported through intraflagellar transport (IFT) complexes out of primary cilia, and eventually, it enters the nucleus and induces the expression of hedgehog target genes (Eguether et al., 2018). Primary cilia, antenna-like organelles, present on most vertebrate cells are responsible for multiple sensory and signaling activities (May, Sroka, & Mick, 2021). The existing results strongly indicate a close relationship between cilia and hedgehog signaling during mechanotransduction.

Similar to the malfunction of hedgehog signaling, mutations in genes encoding for IFT20 or IFT88, two of the most important structural proteins in cilia, cause craniofacial abnormalities (Kitamura et al., 2020, Noda et al., 2016). For example, structural mutations of cilia caused by the depletion of Kif3a or IFT88 in chondrocytes lead to abnormal hedgehog signaling and skeletal abnormalities (Chang et al., 2012, Kinumatsu et al., 2011). Recent studies have shown that the primary cilia membrane contains a variety of ion channels that can convert extracellular mechanical signals into intracellular chemical signals through various signal transduction pathways (Anvarian et al., 2019, Corrigan et al., 2018). Polycystin 2, a nonselective, calcium-permeant, transient receptor potential cation channel, is also expressed on primary cilia (Liu et al., 2018). Moreover, being a mechanoreceptor, mutations in polycystin 2 have been linked to  deformity of temporomandibular joints and abnormal skull shapes (Khonsari et al., 2013); thus, strongly indicating that polycystin 2 plays a vital role in mechanotransduction during craniofacial development.

Considering the similar regulatory role of primary cilia, polycystin 2, and hedgehog signaling in craniofacial development, we envisioned the possible similar effect among them during mechanotransduction of mandibular condylar chondrocytes. Toward this goal, we first evaluated the effect of cyclic tensile strain on the activation of hedgehog signaling in mandibular condylar chondrocytes. We further evaluated the effects of lentiviral vector-mediated IFT88 overexpression and small interfering RNA (siRNA)-mediated IFT88 knockdown on the activation of hedgehog signaling in mandibular condylar chondrocytes. Then, the effects of siRNA-mediated polycystin 2 knockdown on the activation of hedgehog signaling in mandibular condylar chondrocytes were also evaluated. Finally, a coimmunoprecipitation (co-IP) assay under cyclic tensile strain was performed to further study the interaction between IFT88 and polycystin 2.