A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation
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Evaluation Summary:
This study is potentially of high significance to a broad audience of scientists working on vascular reactivity and the role of ion channels in controlling endothelial cell signaling and vessel contractility. The study uses novel Endothelial cell specific knockout mice of Polycystin-1 and 2 (PC1 and PC2) proteins to show the requirement of PC1 and PC2 in flow-mediated vasodilation, how PC-1 and PC-2 interact and that their functions are interdependent. The findings from this study are novel and significant. The animal models used in this study are state of the art and the data overall are of high quality. However, additional data are needed to support the conclusions of the study. Further, additional controls and clarifications are required.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout ( Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca 2+ -dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca 2+ -activated K + channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 ( Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 ( Pkd1 / Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.
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Author Response:
Reviewer #2 (Public Review):
This work aimed to advance knowledge of the roles of polycystin-1 and polycystin-2 (PC-1, PC-2) in the vascular endothelium. For this, the authors developed tamoxifen-inducible Cre-lox models to delete PC-1, PC-2 or both specifically in endothelial cells of mice. Evidence is presented that flow or sheer stress activates PC-1-dependent current in endothelial cells, which is associated with NOS and KCa channel activation, smooth muscle hyperpolarization, and flow-dependent vasodilation. The Jaggar laboratory has recently reported that deletion of endothelial PC-2, a member of the TRP family, leads to loss of flow-induced Ca2+ influx, NOS and SK/IK activation, reduced vasodilation, and higher blood pressure. Thus, the novelty of the current work is the finding that PC-1 is similarly critical …
Author Response:
Reviewer #2 (Public Review):
This work aimed to advance knowledge of the roles of polycystin-1 and polycystin-2 (PC-1, PC-2) in the vascular endothelium. For this, the authors developed tamoxifen-inducible Cre-lox models to delete PC-1, PC-2 or both specifically in endothelial cells of mice. Evidence is presented that flow or sheer stress activates PC-1-dependent current in endothelial cells, which is associated with NOS and KCa channel activation, smooth muscle hyperpolarization, and flow-dependent vasodilation. The Jaggar laboratory has recently reported that deletion of endothelial PC-2, a member of the TRP family, leads to loss of flow-induced Ca2+ influx, NOS and SK/IK activation, reduced vasodilation, and higher blood pressure. Thus, the novelty of the current work is the finding that PC-1 is similarly critical for activation of this pathway by flow, and that it is a physical interaction between membrane-localized PC-1 and PC-2 that underlies complex activation by flow.
Strengths of the current study include the use of powerful inducible knockout models in combination with a wide array of in vivo and ex vivo methods to test hypotheses. Thus, conclusions are based on multiple approaches and are mostly well supported. However, there are some concerns, specifically related to a lack of clarity on the interactions and purported interdependence between PC-1 and PC-2 that warrant further consideration.
1.The prospective impact of the current study is based on the suggestion that interactions between PC-1 and PC-2 via coiled-coil domains are required for activation of inward current by flow. However, the authors did not show evidence, via fluorescence imaging or otherwise (e.g., coIP), that peptides generated to disrupt this interaction actually do so. Does treatment with the coiled-coil domain peptides cause a shift in the PC-1-to-PC-2 distance (using TIRF-SMLM as in Fig 5)?
We have performed new experiments and now show that scrambled peptides of either the PC-1 or PC-2 coiled-coil domains do not alter flow-activated I_Cat in endothelial cells (Figure 6E-G, Figure 6 – figure supplement 2). In contrast, peptides corresponding to the coiled-coil domains present in PC-1 or PC-2 similarly inhibit flow-activated cation currents in endothelial cells.
Multiple different domains in PC-1 and PC-2 physically interact to form the heteromeric complex. Several groups have demonstrated that PC-1 and PC-2 couple via their C-terminal coiled-coils (Qian et al, Nat. Genet. 1997; Zhu et al., PNAS 2011; Yu et al., PNAS 2009; Tsiokas et al., PNAS 1997). Recombinant PC-1 and PC-2 lacking coiled-coils also interact via N-terminal loops (Babich et al, JBC 2004; Feng et al., JBC 2008). The structure of a PC-1/PC-2 heterotetramer that lacked N- and Ctermini was resolved using cryo-EM and indicated that a region between TM6 and TM11 of PC-1 interdigitates with PC-2 (Su et al, Science 2018). As such, it is unlikely that that the coiled-coil domain peptides physically separate PC-1 and PC-2 subunits. Rather, these data suggest that coiled-coil domain coupling in PC-1 and PC-2 is required for flow to activate non-selective cation currents in endothelial cells. In response to your comment, we have expanded discussion of this point in the manuscript.
2.The use of immunoFRET to test for PC-1/PC-2 proximity is not ideal. At minimum, proper negative controls (e.g., use of cells from KO models) should be provided to demonstrate the specificity of this technique for PC-1/PC-2 interactions in endothelial cells.
We agree. As suggested, we have performed new experiments and now provide immunoFRET data for Pkd1 ecKO and Pkd2 ecKO endothelial cells (Figure 4B, C; Figure 4 - figure supplement 1A, B). These data show that N-FRET between PC-1 and PC-2 antibodies is extremely low in Pkd1 ecKO and Pkd2 ecKO endothelial cells.
3.The authors conclude that PC-1/PC-2 clusters in KO cells in SMLM experiments are likely due to non-specific antibody binding. While I agree with this, it raises a question as to the meaning of cluster size data. Considering that the approach relies on fluorophore-tagged antibodies, which cannot be assumed to be in 1:1 stoichiometry with proteins of interest, how relevant is cluster size?
This is an interesting question. All immunofluorescence techniques rely on the use of antibodies to tag proteins. We recognize that the size of clusters reflects the size of both the proteins and antibodies. We now include text in the Discussion stating that the size of the PC-1 and PC-2 clusters reported is the size of both the proteins and the antibodies.
4.Based on data shown in Figure 1, the authors conclude that there is a reduction in inward current with flow. Since the applied technique measures total current, couldn't this result also reflect an increase in outward current (e.g., K+) due to flow that depends on the presence of Ca2+? Also related to these data, the magnitude of initial flow-induced transient current was quite variable (~8 - ~45 pA). Was this due to differences in cell size? The authors should consider expressing data from current recordings in terms of density (pA/pF).
We agree that presenting data as current density is useful and now do so throughout the manuscript, including in figure 1. The results in figure 1 do reflect a flow-activated increase in K^+ current as this response is partially inhibited by apamin/tram-34 (Mackay et al. eLife 2020). We state that there is “a reduction in inward current” as the entire current range in these experiments is negative of 0 pA.
5.Endothelium-specific deletion of PC-1 increased blood pressure, implying that the proposed role for PC-1 is generally applicable to the resistance arterial network; yet here, only small mesenteric vessels were studied. Given the known heterogeneity in the regulation of vascular tone by sheer stress among different arterial beds, is the identified role of PC-1 observed outside of the mesenteric circulation?
We agree that the blood pressure phenotype in Pkd1 ecKO mice suggest that flowactivates PC-1 in endothelial cells of other vascular beds to induce vasodilation. We have now discussed this concept in the manuscript.
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Evaluation Summary:
This study is potentially of high significance to a broad audience of scientists working on vascular reactivity and the role of ion channels in controlling endothelial cell signaling and vessel contractility. The study uses novel Endothelial cell specific knockout mice of Polycystin-1 and 2 (PC1 and PC2) proteins to show the requirement of PC1 and PC2 in flow-mediated vasodilation, how PC-1 and PC-2 interact and that their functions are interdependent. The findings from this study are novel and significant. The animal models used in this study are state of the art and the data overall are of high quality. However, additional data are needed to support the conclusions of the study. Further, additional controls and clarifications are required.
(This preprint has been reviewed by eLife. We include the public reviews …
Evaluation Summary:
This study is potentially of high significance to a broad audience of scientists working on vascular reactivity and the role of ion channels in controlling endothelial cell signaling and vessel contractility. The study uses novel Endothelial cell specific knockout mice of Polycystin-1 and 2 (PC1 and PC2) proteins to show the requirement of PC1 and PC2 in flow-mediated vasodilation, how PC-1 and PC-2 interact and that their functions are interdependent. The findings from this study are novel and significant. The animal models used in this study are state of the art and the data overall are of high quality. However, additional data are needed to support the conclusions of the study. Further, additional controls and clarifications are required.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this report, Mackay et al used Endothelial Cell (EC) specific knockout mice for Polycystin-1 and 2 (PC1 and PC2) proteins to study the function of these proteins in vascular function. PC-1 is a receptor like protein while PC-2 is a TRP family member of mostly non-selective cation channels. They report that either single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 similarly attenuates flow-mediated vasodilation, suggesting that these proteins work together to control vascular function. EC-specific PC-1 knockout mice have increased systemic blood pressure. Using pharmacological compounds, they propose that the vasodilatory function of PC-1 in ECs is mainly mediated by the Ca2+-dependent activation of eNOS, with a small contribution from SK channels. Using coimmunoprecipitations, FRET and …
Reviewer #1 (Public Review):
In this report, Mackay et al used Endothelial Cell (EC) specific knockout mice for Polycystin-1 and 2 (PC1 and PC2) proteins to study the function of these proteins in vascular function. PC-1 is a receptor like protein while PC-2 is a TRP family member of mostly non-selective cation channels. They report that either single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 similarly attenuates flow-mediated vasodilation, suggesting that these proteins work together to control vascular function. EC-specific PC-1 knockout mice have increased systemic blood pressure. Using pharmacological compounds, they propose that the vasodilatory function of PC-1 in ECs is mainly mediated by the Ca2+-dependent activation of eNOS, with a small contribution from SK channels. Using coimmunoprecipitations, FRET and SMLM microscopy, they show that PC-1 and PC-2 form close interactions in the plasma membrane and that knockout of either PC-1 or PC-2 inhibits surface clusters of both PC-1 and PC-2 in ECs. Non-selective cation currents activated by flow in ECs were inhibited by either PC-1 or PC-2 knockout or by C-terminal peptides of PC-1 or PC-2. The authors conclude that endothelial PC-1/PC-2 complexes control arterial contractility through Ca2+-dependent activation of eNOS and SK channels. However, neither increases in intracellular Ca2+ concentrations nor NO production were directly measured in the study. Further, there is a disconnect between current measurements under the physiological conditions of Fig 1 and those of Fig 6 that require clarifications.
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Reviewer #2 (Public Review):
This work aimed to advance knowledge of the roles of polycystin-1 and polycystin-2 (PC-1, PC-2) in the vascular endothelium. For this, the authors developed tamoxifen-inducible Cre-lox models to delete PC-1, PC-2 or both specifically in endothelial cells of mice. Evidence is presented that flow or sheer stress activates PC-1-dependent current in endothelial cells, which is associated with NOS and KCa channel activation, smooth muscle hyperpolarization, and flow-dependent vasodilation. The Jaggar laboratory has recently reported that deletion of endothelial PC-2, a member of the TRP family, leads to loss of flow-induced Ca2+ influx, NOS and SK/IK activation, reduced vasodilation, and higher blood pressure. Thus, the novelty of the current work is the finding that PC-1 is similarly critical for activation of …
Reviewer #2 (Public Review):
This work aimed to advance knowledge of the roles of polycystin-1 and polycystin-2 (PC-1, PC-2) in the vascular endothelium. For this, the authors developed tamoxifen-inducible Cre-lox models to delete PC-1, PC-2 or both specifically in endothelial cells of mice. Evidence is presented that flow or sheer stress activates PC-1-dependent current in endothelial cells, which is associated with NOS and KCa channel activation, smooth muscle hyperpolarization, and flow-dependent vasodilation. The Jaggar laboratory has recently reported that deletion of endothelial PC-2, a member of the TRP family, leads to loss of flow-induced Ca2+ influx, NOS and SK/IK activation, reduced vasodilation, and higher blood pressure. Thus, the novelty of the current work is the finding that PC-1 is similarly critical for activation of this pathway by flow, and that it is a physical interaction between membrane-localized PC-1 and PC-2 that underlies complex activation by flow.
Strengths of the current study include the use of powerful inducible knockout models in combination with a wide array of in vivo and ex vivo methods to test hypotheses. Thus, conclusions are based on multiple approaches and are mostly well supported. However, there are some concerns, specifically related to a lack of clarity on the interactions and purported interdependence between PC-1 and PC-2 that warrant further consideration.
1. The prospective impact of the current study is based on the suggestion that interactions between PC-1 and PC-2 via coiled-coil domains are required for activation of inward current by flow. However, the authors did not show evidence, via fluorescence imaging or otherwise (e.g., coIP), that peptides generated to disrupt this interaction actually do so. Does treatment with the coiled-coil domain peptides cause a shift in the PC-1-to-PC-2 distance (using TIRF-SMLM as in Fig 5)?
2. The use of immunoFRET to test for PC-1/PC-2 proximity is not ideal. At minimum, proper negative controls (e.g., use of cells from KO models) should be provided to demonstrate the specificity of this technique for PC-1/PC-2 interactions in endothelial cells.
3. The authors conclude that PC-1/PC-2 clusters in KO cells in SMLM experiments are likely due to non-specific antibody binding. While I agree with this, it raises a question as to the meaning of cluster size data. Considering that the approach relies on fluorophore-tagged antibodies, which cannot be assumed to be in 1:1 stoichiometry with proteins of interest, how relevant is cluster size?
4. Based on data shown in Figure 1, the authors conclude that there is a reduction in inward current with flow. Since the applied technique measures total current, couldn't this result also reflect an increase in outward current (e.g., K+) due to flow that depends on the presence of Ca2+? Also related to these data, the magnitude of initial flow-induced transient current was quite variable (~8 - ~45 pA). Was this due to differences in cell size? The authors should consider expressing data from current recordings in terms of density (pA/pF).
5. Endothelium-specific deletion of PC-1 increased blood pressure, implying that the proposed role for PC-1 is generally applicable to the resistance arterial network; yet here, only small mesenteric vessels were studied. Given the known heterogeneity in the regulation of vascular tone by sheer stress among different arterial beds, is the identified role of PC-1 observed outside of the mesenteric circulation?
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Reviewer #3 (Public Review):
This study examined the role played by polycystin-1 (PC-1) in endothelium-dependent, flow-mediated vasodilation using a tomixifen-inducible knockout (KO) of endothelial cell PC-1 in mouse mesenteric resistance arteries. To that end, the authors show that a substantial portion of flow-induced: currents in isolated endothelial cells, hyperpolarization in pressurized arteries and vasodilation is attenuated by PC-1 KO, to a similar extent as with endothelial cell KO of polycystin-2 (PC-2). Immuniprecipitation and super-resolution immune localization provided compelling evidence of interactions of clusters of PC-1 and PC-2 that is required for their function in flow-mediated vasodilation.
Strengths of this study include the combination of approaches utilized including the use of endothelial specific KO's of PC-1 …
Reviewer #3 (Public Review):
This study examined the role played by polycystin-1 (PC-1) in endothelium-dependent, flow-mediated vasodilation using a tomixifen-inducible knockout (KO) of endothelial cell PC-1 in mouse mesenteric resistance arteries. To that end, the authors show that a substantial portion of flow-induced: currents in isolated endothelial cells, hyperpolarization in pressurized arteries and vasodilation is attenuated by PC-1 KO, to a similar extent as with endothelial cell KO of polycystin-2 (PC-2). Immuniprecipitation and super-resolution immune localization provided compelling evidence of interactions of clusters of PC-1 and PC-2 that is required for their function in flow-mediated vasodilation.
Strengths of this study include the combination of approaches utilized including the use of endothelial specific KO's of PC-1 and PC-2, patch-clamp of isolated mesenteric endothelial cells, pressure myography, and sharp micro electrode measurement of membrane potential in pressurized arteries to the functional role of PC-1 and PC-2, along with immunoprecipitation, N-FRET, and super-resolution microscopy to show Interactions between PC-1 and PC-2. Finally, their use of competing peptides to provide additional evidence of physical interactions of PC-1 and PC-2 required for flow-mediated endothelial cell action currents was an additional strength.
Weaknesses that detracted from the strengths of this study include the following. First, the use of only mesenteric arteries in this study, such that the generality of their findings remain to be established. Second, while the authors clearly show roles for PC-1 and PC-2 in flow mediated vasodilation, how these proteins sense flow (shear stress) (is it direct or linked to some other "sensor") remains to be established and was not addressed by the authors. Finally, several other endothelial cell ion channels have been proposed to play roles in flow-mediated vasodilation, but their role and how they "fit" into an integrated scheme with PC-1 and PC-2 remains to be established.
Despite these weaknesses, this study moves the field forward and should provide ample impetus for further investigation.
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