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Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Miura S, Sato K, Kato-Negishi M, Teshima T, Takeuchi S - Nat Commun (2015)

Bottom Line: Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells.We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation.TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS.

View Article: PubMed Central - PubMed

Affiliation: Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

ABSTRACT
Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

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Involvement of TRPV6 in FSS-induced Ca2+ entry in BeWo cells.(a) Expression of TRPV ion channels in BeWo cells was analysed by RT–PCR. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. (b) TRPV6 knockdown was evaluated by immunoblot analysis using anti-TRPV6 antibody and anti-β-actin antibody (loading control). (c,d) Time course of Fura-2 fluorescence in the TRPV6 knockdown BeWo cells. Fluid flow (5 μl min−1) was applied from t=3 min onward, and calcium imaging was performed at the centre area of the chamber. Data shown represent the Fura-2 ratio (F340/F380) and mean±s.d. (n=33). The FSS-induced increase of the Fura-2 ratio (maximal Fura-2 ratio−basal Fura-2 ratio (t=3 min)) in c is shown as the percentage relative to the control (d). **P<0.01, Student's t-test.
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f5: Involvement of TRPV6 in FSS-induced Ca2+ entry in BeWo cells.(a) Expression of TRPV ion channels in BeWo cells was analysed by RT–PCR. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. (b) TRPV6 knockdown was evaluated by immunoblot analysis using anti-TRPV6 antibody and anti-β-actin antibody (loading control). (c,d) Time course of Fura-2 fluorescence in the TRPV6 knockdown BeWo cells. Fluid flow (5 μl min−1) was applied from t=3 min onward, and calcium imaging was performed at the centre area of the chamber. Data shown represent the Fura-2 ratio (F340/F380) and mean±s.d. (n=33). The FSS-induced increase of the Fura-2 ratio (maximal Fura-2 ratio−basal Fura-2 ratio (t=3 min)) in c is shown as the percentage relative to the control (d). **P<0.01, Student's t-test.

Mentions: Microvilli contain various mechanosensitive cation channels including TRP protein family, which are activated by a wide range of physciochemical stimuli1617. Although some subfamilies of TRP channels are capable of a transduction of mechanical signals into the increase of cytosolic calcium, it has been reported that TRPV5 and TRPV6, both of which are TRPV subfamily members, are Ca2+-selective ion channels that play a pivotal role in the calcium absorption in epithelial tissues, including the intestine, kidney and placenta181920. In our preliminary experiment, capsaicin, which is known to activate TRPV channels2122, induced microvilli in the immunocytochemical detection of Ezrin, as well as Ezrin mRNA levels (approximately twofold at 1 μM capsaicin compared with the control) in BeWo cells cultured under static conditions (Supplementary Fig. 2; Ezrin is a microvilli-localizing protein that functions as a linker between plasma membrane and microvillous actin cytoskeleton23). Moreover, addition of a general inhibitor of receptor-operated calcium entry, SKF96365, resulted in the partial inhibition of microvilli formation visualized by Ezrin (Supplementary Fig. 3). On the basis of these facts, we first investigated the expression of TRPV subfamily, which consists of six homologous members, in BeWo cells by reverse transcription PCR (RT–PCR; Fig. 5a). BeWo cells expressed TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6. Above all, TRPV6 was reported to be activated by FSS24, which indicates that TRPV6 is a candidate responsible for FSS-induced Ca2+ influx in BeWo cells.


Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6.

Miura S, Sato K, Kato-Negishi M, Teshima T, Takeuchi S - Nat Commun (2015)

Involvement of TRPV6 in FSS-induced Ca2+ entry in BeWo cells.(a) Expression of TRPV ion channels in BeWo cells was analysed by RT–PCR. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. (b) TRPV6 knockdown was evaluated by immunoblot analysis using anti-TRPV6 antibody and anti-β-actin antibody (loading control). (c,d) Time course of Fura-2 fluorescence in the TRPV6 knockdown BeWo cells. Fluid flow (5 μl min−1) was applied from t=3 min onward, and calcium imaging was performed at the centre area of the chamber. Data shown represent the Fura-2 ratio (F340/F380) and mean±s.d. (n=33). The FSS-induced increase of the Fura-2 ratio (maximal Fura-2 ratio−basal Fura-2 ratio (t=3 min)) in c is shown as the percentage relative to the control (d). **P<0.01, Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660203&req=5

f5: Involvement of TRPV6 in FSS-induced Ca2+ entry in BeWo cells.(a) Expression of TRPV ion channels in BeWo cells was analysed by RT–PCR. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. (b) TRPV6 knockdown was evaluated by immunoblot analysis using anti-TRPV6 antibody and anti-β-actin antibody (loading control). (c,d) Time course of Fura-2 fluorescence in the TRPV6 knockdown BeWo cells. Fluid flow (5 μl min−1) was applied from t=3 min onward, and calcium imaging was performed at the centre area of the chamber. Data shown represent the Fura-2 ratio (F340/F380) and mean±s.d. (n=33). The FSS-induced increase of the Fura-2 ratio (maximal Fura-2 ratio−basal Fura-2 ratio (t=3 min)) in c is shown as the percentage relative to the control (d). **P<0.01, Student's t-test.
Mentions: Microvilli contain various mechanosensitive cation channels including TRP protein family, which are activated by a wide range of physciochemical stimuli1617. Although some subfamilies of TRP channels are capable of a transduction of mechanical signals into the increase of cytosolic calcium, it has been reported that TRPV5 and TRPV6, both of which are TRPV subfamily members, are Ca2+-selective ion channels that play a pivotal role in the calcium absorption in epithelial tissues, including the intestine, kidney and placenta181920. In our preliminary experiment, capsaicin, which is known to activate TRPV channels2122, induced microvilli in the immunocytochemical detection of Ezrin, as well as Ezrin mRNA levels (approximately twofold at 1 μM capsaicin compared with the control) in BeWo cells cultured under static conditions (Supplementary Fig. 2; Ezrin is a microvilli-localizing protein that functions as a linker between plasma membrane and microvillous actin cytoskeleton23). Moreover, addition of a general inhibitor of receptor-operated calcium entry, SKF96365, resulted in the partial inhibition of microvilli formation visualized by Ezrin (Supplementary Fig. 3). On the basis of these facts, we first investigated the expression of TRPV subfamily, which consists of six homologous members, in BeWo cells by reverse transcription PCR (RT–PCR; Fig. 5a). BeWo cells expressed TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6. Above all, TRPV6 was reported to be activated by FSS24, which indicates that TRPV6 is a candidate responsible for FSS-induced Ca2+ influx in BeWo cells.

Bottom Line: Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells.We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation.TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS.

View Article: PubMed Central - PubMed

Affiliation: Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

ABSTRACT
Microvilli are cellular membrane protrusions present on differentiated epithelial cells, which can sense and interact with the surrounding fluid environment. Biochemical and genetic approaches have identified a set of factors involved in microvilli formation; however, the underlying extrinsic regulatory mechanism of microvilli formation remains largely unknown. Here we demonstrate that fluid shear stress (FSS), an external mechanical cue, serves as a trigger for microvilli formation in human placental trophoblastic cells. We further reveal that the transient receptor potential, vanilloid family type-6 (TRPV6) calcium ion channel plays a critical role in flow-induced Ca(2+) influx and microvilli formation. TRPV6 regulates phosphorylation of Ezrin via a Ca(2+)-dependent phosphorylation of Akt; this molecular event is necessary for microvillar localization of Ezrin in response to FSS. Our findings provide molecular insight into the microvilli-mediated mechanoresponsive cellular functions, such as epithelial absorption, signal perception and mechanotransduction.

Show MeSH
Related in: MedlinePlus