Osteocytes project long, slender processes throughout the mineralized matrix of bone where they connect and communicate with effector cells. The interconnected cellular projections form the functional lacuno-canalicular system (LCS) that allows fluid to pass for cell to cell communication and nutrient and waste exchange. Osteocytes are the most abundant cells in bone and are essential for mechanosensation and transduction of signals to regulate processes such as bone formation and resorption. The focus of this work was to elucidate the influence of the large heparan sulfate proteoglycan perlecan/HSPG2 (PLN) on the ultrastructural properties of the osteocyte LCS. Additionally, the role of voltage sensitive calcium channels (VSCCs) in osteocyte mechanotransduction was investigated, and studies exploring the ability of these channels to form complexes with extracellular matrix (ECM) molecules were undertaken.

Prevention of bone mineral intrusion into the pericellular space of the lacuno-canalicular pericellular space is crucial to maintain unimpeded interstitial fluid movement. Factors that prevent the clear pericellular space of the LCS and keep it open to fluid movement remain unclear. Because of their activity in inhibiting hydroxyapatite formation, heparan sulfate containing macromolecules are viable candidates to perform this function.

In this study, along with my colleagues, I examined osteocyte lacuno-canalicular morphology in mice deficient in the large heparan sulfate proteoglycan (HSPG) PLN in this tissue. Immunohistochemical imaging demonstrates PLN expression localized to the osteocyte LCS of cortical bone and electron micrograph immunogold data reveal the presence of this proteoglycan (PG) in the pericellular space of the LCS. In contrast, PLN expression was severely decreased in cortical bone of PLN deficient mice. Ultrastructural measurements using electron micrograph images of PLN deficient mice demonstrate a significant decrease in osteocyte canalicular pericellular area, resulting from a reduction in the total canalicular area, when compared to controls. Additionally, PLN deficient mice show significantly diminished canalicular density and a significant reduction in the number of transverse tethering elements per canaliculus.

These data indicate that PLN is present in the pericellular space of the osteocytic processes in the LCS in a location where it could function to help maintain clear fluid path. Additionally, as shown with PLN deficient mice, PLN contributes to the integrity of the osteocyte LCS by maintaining the size of the pericellular space, an essential task to promote uninhibited interstitial fluid movement in this mechanosensitive environment. This work thus identifies a new barrier function for PLN in murine cortical bone.

Regulation of skeletal remodeling is the focus of great attention in translational osteoporosis research. A recent linkage association study identified CACNA2D2, which encodes for an α₂δ subunit within the VSCC complex, as a novel susceptibility marker for bone mineral density (BMD) variation. VSCCs are a functional complex of polypeptide units consisting of a pore forming α₁ subunit, an intracellular β subunit, disulfide linked α₂ and δ subunits, which form an extracellular auxiliary complex and a γ subunit in some tissues.

VSCCs play an important role in osteoblast mechanotransduction;​ yet, their role in osteocyte function remains elusive. Osteoblasts predominantly express L-type Ca_v1.2 VSCCs;​ however, osteocytes express the T-type Ca_v3.2 subunit. The presence of auxiliary VSCC subunits in osteocytes has not been described and the ability of these subunits to associate with T-type VSCCs has not been shown in bone or any other tissue. T-type VSCCs have lower conductances than L-type channels, a property that can impact osteocyte physiology. The purpose of this study was to measure expression of VSCC subunits in osteocytes, to determine which auxiliary subunits associate with T-type VSCCs in these cells, and to investigate the role of the α₂δ₁ auxiliary subunit in mechanosensation.

RT-PCR, Western blot and immunostaining were used to measure expression levels of VSCC subunits in osteocytic MLO-Y4 cells. Coimmunoprecipitation (co-IP) demonstrated an association of the α₂δ₁ subunit with the Cav3.2 subunit in MLO-Y4 cells. Expression of all known VSCC subunits was assessed in the MLO-Y4 cell line. Along with my colleagues, I demonstrated that osteocytes expressed the T-type Cav3.2 subunit to a much higher extent than the L-type Cav1.2 subunit which was nearly undetectable in well-differentiated cells. The α₂δ₁, β_1-3 and γ₇ subunit transcripts were also present within osteocytic cells. Co-IP revealed the ability of the α₂δ₁ subunit to complex with the Cav3.2 subunit suggesting that these subunits associate in the MLO-Y4 cell line. In the absence of the α₂δ₁ subunit stretchinduced ATP release was significantly decreased from MLO-Y4 osteocyte-like cells. The diminished ATP release in cells lacking the α₂δ₁ subunit was attributed to α₂δ₁-mediated trafficking of the Ca_v3.2 (α_1H) subunit to the plasma membrane of osteocytic cells.

Additional studies provide preliminary data to support the role for the α₂δ₁ subunit to modulate Ca²⁺ permeability through T-type Ca_v3.2 (α_1H) VSCCs in osteocytes. Recent evidence indicates that the α₂δ subunit interacts with the ECM, providing a mechanism through which the conductance of these channels may be modulated by mechanical stimuli. Association of α₂δ₁ with T-type channels in osteocytes can stabilize the channel, providing a mechanism for interaction with the extracellular environment. Immunocytochemistry assays provide a potential role for the HSPG PLN to complex with VSCC subunits, revealing a possible regulatory mechanism initiated through the ECM.

This protein complex may provide a crucial function for mechanosensitive osteocytes, having broad implications in skeletal remodeling and bone related pathologies. Bone pathologies, especially those related to diminished bone density, are a tremendous source of disability and morbidity among the elderly population. The studies presented in this work provide several novel contributions for the role of PLN and VSCCs in osteocyte physiology which have excellent potential to forge new clinically-centered discoveries, providing enhanced treatments for persons with bone pathologies.