In this study, we directed a FRET-based Ca2+ biosensor to the primary cilium and loaded cells with a nontargeted fluorescent Ca2+ indicator dye to resolve the local Ca2+ environment in the osteocyte primary cilium from the cytosol. We monitored ciliary
and cytosolic Ca2+ levels using an epifluorescence microscopy system and observed flow-induced Ca2+ peaks in both domains. Trpv4 deficiency reduced flow-induced ciliary Ca2+ peaks but did not impair flow-induced cytosolic Ca2+ mobilization, illustrating that the primary cilium microdomain is distinct from the
cytosol. Thapsigargin treatment impaired flow-induced ciliary and cytosolic Ca2+ peaks, demonstrating that intracellular Ca2+ release and separate Ca2+ entry through TRPV4 are both components of ciliary Ca2+ mobilization. In contrast, knockdown of Pkd2 and Piezo1 did not affect ciliary or cytosolic Ca2+ peaks. Last, we linked the role of TRPV4 in regulating flow-induced ciliary Ca2+ mobilization with a downstream osteogenic response at the transcriptional level by
determining that flow-induced changes in Cox-2 expression depend on TRPV4. Collectively, our study demonstrates that the loading-induced
ciliary Ca2+ mechanism is different between kidney epithelia and osteocytes.
After observing flow-induced Ca2+ peaks in both the osteocyte primary cilium and cytosol, we were motivated to identify
the source of the ciliary Ca2+ peak. To deplete intracellular Ca2+ stores, we treated cells with thapsigargin and continued to observe ciliary Ca2+ peaks, suggesting that Ca2+-permeable channels on the primary cilium have a role in mediating flow-induced Ca2+ entry. However, thapsigargin treatment did lower flow-induced ciliary and cytosolic
Ca2+ peak magnitudes and responsiveness compared with untreated cells, indicating that
intracellular Ca2+ release is a component of flow-induced ciliary Ca2+ mobilization in osteocytes. Furthermore, a statistically significant delay in ciliary
and cytosolic Ca2+ peaks occurred in thapsigargin-treated cells compared with controls. Thus, our data
demonstrate that intracellular Ca2+ release contributes, in part, to the local primary cilia Ca2+ environment and suggests that the primary cilium serves as an important signal integrator.
It is important to note that a general ensemble analysis of our data did not reveal
an effect of PC2, TRPV4, or PIEZO1 on flow-induced ciliary Ca2+ mobilization. In fact, the only significant reduction in peak Ca2+ mobilization was demonstrated in the cytosol with thapsigargin treatment. On the
one hand, although the effect of thapsigargin on flow-induced Ca2+ mobilization was clear, using such a potent agent provides limited details. As expected,
extracting the finer details of the role of Ca2 channels in targeted Ca2 channel blocking experiments required more selective analysis, which included categorizing
cells into responders and nonresponders. While selecting a discriminating threshold
can be confounding to the analysis, we found no significant difference in average
peak ciliary Ca2+ mobilization with any treatment, suggesting that the results of this approach are
not sensitive to the magnitude of the threshold (Additional file 5: Figure S4).
In this study, we present evidence that fluid flow activates TRPV4 on the primary
cilium membrane and mediates Ca2+ influx. Using immunocytochemistry techniques, we determined that the stretch-activated
Ca2+-permeable channel TRPV4 localizes to the primary cilium and plasma membrane. Interestingly,
TRPV4 (and PC2 and PIEZO1) appears throughout the cell, which is consistent with TRPV4
and PC2 immunostaining in the literature 32]-35]. While the distribution of the channels appear higher on the cell membrane relative
to the primary cilium and may mediate a larger Ca2+ flux compared with the flux into the primary cilium, it is likely that the machinery
and spatiotemporal molecular pathways unique to the primary cilium play a role in
downstream mechanotransduction. Our flow experiments revealed that Trpv4 knockdown lowered flow-induced ciliary Ca2+ peaks but did not impair cytosolic Ca2+ peaks. Unlike kidney epithelia, where Ca2+ reportedly enters the primary cilium through PC2 and induces CICR via ryanodine receptor
activation 18], Trpv4 deficiency in osteocytes did not affect cytosolic Ca2+ mobilization. This result suggests that the TRPV4-mediated ciliary Ca2+ microdomain does not regulate CICR in osteocytes. This is different from astrocytes,
where TRPV4-mediated CICR regulates neurovascular coupling in an IP3R-regulated system 36]. It is also possible that other mechanically activated Ca2+-permeable channels compensate for the loss of TRPV4 function in the cytosol, which
is consistent with data from this study showing that the loss of Piezo1 may sensitize cells to flow. Knockdown of PC2, TRPV4, and PIEZO1 channels did not
impair the flow-induced cytosolic Ca2+ response, which suggests that a different mechanism is in play that maintains normal
cytosolic Ca2+ levels. Taken together, TRPV4’s location in an area of high membrane strain on the
primary cilium and dependence of the flow-induced ciliary Ca2+ peak on TRPV4 suggest that the primary cilium acts as a Ca2+ and mechanical signaling nexus dependent on TRPV4.
Consistent with these results, previously, Malone et al. blocked flow-induced extracellular
Ca2+ entry into MC3T3 osteoblasts using gadolinium chloride, which did not eliminate the
flow-induced cytosolic Ca2+ flux 14]. In addition to demonstrating that flow-induced cytosolic Ca2+ flux is not dependent on extracellular Ca2+ entry through stretch-activated channels, Malone et al. also reported that the removal
of primary cilia did not affect flow-induced cytosolic Ca2+ flux in osteocytes. The results in this study, here, are consistent with the previous
paper and provide additional insight to the understanding of osteocyte mechanotransduction.
Using advanced imaging techniques that provide enhanced resolution, our data demonstrate
that flow-induced cytosolic Ca2+ flux is independent of extracellular Ca2+ entry through stretch-activated channels; furthermore, flow-induced ciliary Ca2+ flux is dependent on the Ca2+-permeable, stretch-activated TRPV4 channel. Thus, our data suggest that Ca2+ mobilization occurs differently in the cytosol versus the primary cilium during osteocyte
mechanotransduction.
Our understanding of primary cilium bending mechanics and mechanical forces on the
plasma membrane covering the primary cilium is essential to elucidating the molecular
mechanism of mechanotransduction mediated by the primary cilium. Recently, Young et
al. studied the tension force distribution along a primary cilium under flow and suggested
that stretch-activated ion channels are likely to be activated and open near the base
of the primary cilium where tension force is the highest 37]. While primary cilia bending is one potential physical event, it is possible that
primary cilium deflection is not physiologic and that mechanical loading of the primary
cilium occurs in other ways. For example, ?-1 integrins are localized to MDCK primary
cilia, and ?-1 integrins have been implicated in mediating osteocyte mechanotransduction
and loading-induced bone formation 38]-40]. Thus, increased membrane tension is not limited to primary cilia bending and may
involve primary cilia integrin-extracellular matrix interactions.
We anticipate that TRPV4 will be an attractive pharmacologic target for treating disuse-induced
bone loss due to its role mediating osteocyte mechanotransduction and its sensitivity
to existing biochemical agents (agonists: 4?-PDD, GSK1016790A, and RN1747 and antagonist:
RN1734) 41]-43]. Thus, treatment with TRPV4 agonists and therapies that elongate osteocyte primary
cilia (lithium chloride, hydrogen sulfide, interleukin-1) to enhance mechanical strain
levels may amplify osteogenic responses and prevent disuse-induced bone loss in patients
restricted to bed rest 8],44],45]. Furthermore, O’Conor et al. have shown that TRPV4 plays a role as a physical transducer
in chondrocytes, which may provide insight into functional cartilage tissue engineering
approaches 46].
Other groups have suggested that TRPV4 is a candidate therapeutic target for bone
loss disease. Mizoguchi et al. and Masuyama et al. have examined TRPV4 deficiency
in unloading-induced bone formation, and they determined that Trpv4 knockout reduces unloading-induced bone loss due to suppressed osteoclast numbers
and impaired bone resorption 47],48]. Interestingly, Mizoguchi et al. do not exclude the possibility that Trpv4 is expressed by osteocytes. Osteocytes are mechanosensing cells in bone that regulate
osteoclasts’ resorption activities, and it will be important to understand the role
of TRPV4 in the population of cells involved in mechanotransduction. The effect of
an osteocyte-specific Trpv4 deletion in loading-induced bone formation in vivo would provide evidence indicating whether TRPV4 is involved in the mechanotransduction
process versus restricted to mediating osteoclast numbers and bone resorption. Additionally,
it is important to recognize that primary cilia play important roles in development;
however, in this study, we have not examined how TRPV4 plays a role in developmental
ciliary pathways such as Hedgehog and Wnt signaling pathways.
While Jin et al. reported that flow-induced Ca2+ mobilization occurs in primary cilia before cytosolic Ca2+ increases in kidney epithelial cells, our flow studies do not demonstrate a clear
difference in the timing between ciliary and cytosolic Ca2+ peaks in osteocytes 18]. While the amount of ciliary Ca2+ peaks occurring before cytosolic Ca2+ peaks is numerically higher than the number of ciliary Ca2+ peaks occurring after cytosolic Ca2+ peaks, it is unclear if ciliary Ca2+ triggers cytosolic Ca2+ increases. Unlike the side-dimension imaging method leveraged by Jin et al. to capture
calcium signaling along the length of the primary cilium, our imaging method collected
signal from only a part of the primary cilium that was in plane. Thus, we are unable
to characterize a relationship between ciliary and cytosolic Ca2+ peak timing.
Several other groups have demonstrated that flow-induced ciliary Ca2+ mobilization is dependent on PC2 in kidney epithelia 15],18],31]. Our data suggest that flow-induced ciliary Ca2+ mobilization is dependent on TRPV4, and not PC2, in osteocytes. We conducted similar
flow studies with kidney epithelial cells to verify that this difference was due to
cell type and not the experimental approach. Our studies with IMCD cells also showed
that flow-induced ciliary and cytosolic Ca2+ increases depend on PC2. The consistency in our results suggests that the mechanism
of mechanotransduction mediated by primary cilia varies across different tissue contexts.
Another difference between the MLO-Y4 and IMCD flow studies was the type of flow regime,
consisting of OFF resulting in a 10-dyn/cm2 shear stress for MLO-Y4 cells and steady flow resulting in a 5-dyn/cm2 shear stress for IMCD cells. Previously, Malone et al. determined that flow-induced
Ca2+ flux differences in MC3T3-E1 osteoblasts and MDCK kidney cells did not depend on
these specific flow regimes, which suggests that primary cilium-mediated mechanosensation
in osteoblasts and kidney cells is indeed different 14]. Thus, the application of different but physiologically relevant mechanical loads
was appropriate for elucidating intricacies in the mechanotransduction mechanism in
IMCD and MLO-Y4 cells.
Interestingly, in addition to being well-established in kidney epithelial mechanotransduction
15],18],31], PC2 has recently been implicated in osteocyte mechanotransduction 49]. The authors found siRNA-mediated knockdown of PC2 inhibited downstream flow-induced
nitric oxide production and inducible nitric oxide synthase. While fluid flow-induced
shear stress is known to activate the nitric oxide pathway, this activation occurs
over a much longer time scale of hours compared to Ca2+ flux of seconds and minutes. Here, we studied the early signaling response to flow
occurring within seconds after exposure to flow. Thus, our finding that PC2 was independent
of mechanically induced ciliary and cytosolic Ca2+ signaling in osteocytes does not contradict the findings by Xu et al. It is possible
that PC2 is involved in the later downstream signaling response to flow. While we
found that PC2 may not be involved in the early Ca2+ response to flow, it is nonetheless an important channel in bone. Pkd2 mutations have been implicated in skeletal development 50],51], and mutations in Pkd1, encoding the other subunit of the polycystin complex, have impaired both skeletal
development and adaptation 52].