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Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells.

Henzi T, Schwaller B - PLoS ONE (2015)

Bottom Line: With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated.Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected.In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

No MeSH data available.


Related in: MedlinePlus

Madin-Darby canine kidney (MDCK) cells stably expressing PV.Lentiviral infection of MDCK cells led to cytoplasmic expression of PV evidenced by PV immunohistochemistry; PV expression levels were variable in clones selected by serial dilutions (A). Relative PV protein expression levels of selected clones were quantified by Western blot analyses (B). Non-transfected (Con) and EGFP-transfected clones (EGFP2) were negative for PV. Expression levels in individual clones were considerably different; e.g. levels in clone PV11 were almost 10-fold lower than in the high-expressing clone PV15. COX1 and ATP synthase subunit β protein levels of clones without PV and clones highly expressing PV (PV15, PV19, PV29) were compared (C). COX1 was significantly reduced in the PV-expressing clones (*p = 0.0099), ATP synthase subunit β levels were not different; p = 0.890823; n = 6 independent experiments, mean ± sem). The mRNA level of COX1 gene coding for COX1 was assessed by qRT-PCR (D). In the PV-expressing PV15 clone, the COX1 signal was decreased by 48%; p = 0.00005). The results are the mean of 2 independent experiments (mean ± sem).
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pone.0142005.g004: Madin-Darby canine kidney (MDCK) cells stably expressing PV.Lentiviral infection of MDCK cells led to cytoplasmic expression of PV evidenced by PV immunohistochemistry; PV expression levels were variable in clones selected by serial dilutions (A). Relative PV protein expression levels of selected clones were quantified by Western blot analyses (B). Non-transfected (Con) and EGFP-transfected clones (EGFP2) were negative for PV. Expression levels in individual clones were considerably different; e.g. levels in clone PV11 were almost 10-fold lower than in the high-expressing clone PV15. COX1 and ATP synthase subunit β protein levels of clones without PV and clones highly expressing PV (PV15, PV19, PV29) were compared (C). COX1 was significantly reduced in the PV-expressing clones (*p = 0.0099), ATP synthase subunit β levels were not different; p = 0.890823; n = 6 independent experiments, mean ± sem). The mRNA level of COX1 gene coding for COX1 was assessed by qRT-PCR (D). In the PV-expressing PV15 clone, the COX1 signal was decreased by 48%; p = 0.00005). The results are the mean of 2 independent experiments (mean ± sem).

Mentions: In fast-twitch muscle and cerebellar Purkinje cells exemplifying excitable cells, an inverse regulation of PV and mitochondria was demonstrated previously [7–9]. However, whether such a regulation also occurs in non-excitable cells, and whether it operates bi-directionally was yet unknown. In initial Western blot experiments we ascertained that no other major Ca2+-binding proteins, acting as putative cytosolic Ca2+ buffers were present in MDCK cells; results for PV, CB D-28k and CB D-9k were all negative. MDCK cells were infected with lentivirus (pLVTHM-PV vector; [25]) stably expressing PV in MDCK cells. From the total of PV-expressing MDCK cell clones characterized by variable expression levels, individual clones were selected by serial dilution, expanded and then screened for PV expression by IHC (Fig 4A). PV expression levels of clones were determined semi-quantitatively by Western blot analysis; non-infected MDCK cells and pLVTHM-EGFP infected MDCK cells served as negative controls (Fig 4B). In order to compare relative PV expression levels, the signal of clone PV15 having the highest PV expression level was defined as 100%. High PV levels were detected in clones PV15, PV19, PV29, clearly lower ones in clones PV2 and PV11 (Fig 4B). No PV signal was detected in control MDCK and in EGFP-lentivirus infected MDCK cells. Since we expected the largest differences to exist between control and high PV-expressing MDCK clones, quantitative expression levels of COX1 and ATP synthase subunit β were determined in the high PV-expression clones PV15, PV19 and PV29 and compared to control MDCK cells. The mean COX1 level of the 3 PV-clones was significantly reduced, while ATP synthase subunit β levels were not affected by the overexpression of PV (Fig 4C). With respect to COX1, this is the inverse of what was observed in DCT of PV-/-mice. qRT-PCR of RNA isolated from clone PV15 revealed the COX1 mRNA coding for COX1 to be down-regulated (Fig 4D). In order to demonstrate the reversibility of the effect, PV expression in pLVTHM PV-infected clone PV15, was silenced by two different approaches: constitutive down-regulation of PV via lentiviral-mediated shRNA and IPTG-inducible down-regulation using the MISSION® Inducible shRNA system. The constitutive method reduced PV expression levels to about 10% of the initial amount evidenced after 10 days of puromycin selection (Fig 5A), while with the inducible system, PV expression levels decreased to 45% after 4 days of IPTG treatment and to less than 40% after 7 days of IPTG treatment (Fig 5B). IHC for PV using a fluorescent secondary antibody confirmed the strong silencing effect of the constitutive lentiviral-mediated shRNA (Fig 5C). As the result of the PV-down-regulation was more robust with the constitutive shRNA expression system, this one was chosen for the determination of COX1 and ATP synthase subunit β levels by Western blot analyses. Lentiviral shRNA-silencing of ectopically expressed PV in MDCK cells led to a significant increase in COX1 compared to the untreated PV-positive cell clone PV15 (Fig 6). Also the mitochondrial volume determined by FACS analysis was increased after Pvalb mRNA silencing, from 65.8 ± 11.5% to 79.2 ± 14.1% (p<0.05). Thus, the changes induced by ectopic PV expression were reverted partially by Pvalb shRNA treatment documenting the bidirectional regulation in an identical setting. The partial reversal of the mitochondrial volume might be the result of the incomplete PV down-regulation by the shRNA approach. ATP synthase subunit β levels were unchanged after PV down-regulation (Fig 6), i.e. neither PV’s up- nor its subsequent down-regulation affects ATP synthase subunit β levels. These experiments clearly demonstrated that PV and COX1 were bi-directionally and inversely regulated in MDCK cells.


Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells.

Henzi T, Schwaller B - PLoS ONE (2015)

Madin-Darby canine kidney (MDCK) cells stably expressing PV.Lentiviral infection of MDCK cells led to cytoplasmic expression of PV evidenced by PV immunohistochemistry; PV expression levels were variable in clones selected by serial dilutions (A). Relative PV protein expression levels of selected clones were quantified by Western blot analyses (B). Non-transfected (Con) and EGFP-transfected clones (EGFP2) were negative for PV. Expression levels in individual clones were considerably different; e.g. levels in clone PV11 were almost 10-fold lower than in the high-expressing clone PV15. COX1 and ATP synthase subunit β protein levels of clones without PV and clones highly expressing PV (PV15, PV19, PV29) were compared (C). COX1 was significantly reduced in the PV-expressing clones (*p = 0.0099), ATP synthase subunit β levels were not different; p = 0.890823; n = 6 independent experiments, mean ± sem). The mRNA level of COX1 gene coding for COX1 was assessed by qRT-PCR (D). In the PV-expressing PV15 clone, the COX1 signal was decreased by 48%; p = 0.00005). The results are the mean of 2 independent experiments (mean ± sem).
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pone.0142005.g004: Madin-Darby canine kidney (MDCK) cells stably expressing PV.Lentiviral infection of MDCK cells led to cytoplasmic expression of PV evidenced by PV immunohistochemistry; PV expression levels were variable in clones selected by serial dilutions (A). Relative PV protein expression levels of selected clones were quantified by Western blot analyses (B). Non-transfected (Con) and EGFP-transfected clones (EGFP2) were negative for PV. Expression levels in individual clones were considerably different; e.g. levels in clone PV11 were almost 10-fold lower than in the high-expressing clone PV15. COX1 and ATP synthase subunit β protein levels of clones without PV and clones highly expressing PV (PV15, PV19, PV29) were compared (C). COX1 was significantly reduced in the PV-expressing clones (*p = 0.0099), ATP synthase subunit β levels were not different; p = 0.890823; n = 6 independent experiments, mean ± sem). The mRNA level of COX1 gene coding for COX1 was assessed by qRT-PCR (D). In the PV-expressing PV15 clone, the COX1 signal was decreased by 48%; p = 0.00005). The results are the mean of 2 independent experiments (mean ± sem).
Mentions: In fast-twitch muscle and cerebellar Purkinje cells exemplifying excitable cells, an inverse regulation of PV and mitochondria was demonstrated previously [7–9]. However, whether such a regulation also occurs in non-excitable cells, and whether it operates bi-directionally was yet unknown. In initial Western blot experiments we ascertained that no other major Ca2+-binding proteins, acting as putative cytosolic Ca2+ buffers were present in MDCK cells; results for PV, CB D-28k and CB D-9k were all negative. MDCK cells were infected with lentivirus (pLVTHM-PV vector; [25]) stably expressing PV in MDCK cells. From the total of PV-expressing MDCK cell clones characterized by variable expression levels, individual clones were selected by serial dilution, expanded and then screened for PV expression by IHC (Fig 4A). PV expression levels of clones were determined semi-quantitatively by Western blot analysis; non-infected MDCK cells and pLVTHM-EGFP infected MDCK cells served as negative controls (Fig 4B). In order to compare relative PV expression levels, the signal of clone PV15 having the highest PV expression level was defined as 100%. High PV levels were detected in clones PV15, PV19, PV29, clearly lower ones in clones PV2 and PV11 (Fig 4B). No PV signal was detected in control MDCK and in EGFP-lentivirus infected MDCK cells. Since we expected the largest differences to exist between control and high PV-expressing MDCK clones, quantitative expression levels of COX1 and ATP synthase subunit β were determined in the high PV-expression clones PV15, PV19 and PV29 and compared to control MDCK cells. The mean COX1 level of the 3 PV-clones was significantly reduced, while ATP synthase subunit β levels were not affected by the overexpression of PV (Fig 4C). With respect to COX1, this is the inverse of what was observed in DCT of PV-/-mice. qRT-PCR of RNA isolated from clone PV15 revealed the COX1 mRNA coding for COX1 to be down-regulated (Fig 4D). In order to demonstrate the reversibility of the effect, PV expression in pLVTHM PV-infected clone PV15, was silenced by two different approaches: constitutive down-regulation of PV via lentiviral-mediated shRNA and IPTG-inducible down-regulation using the MISSION® Inducible shRNA system. The constitutive method reduced PV expression levels to about 10% of the initial amount evidenced after 10 days of puromycin selection (Fig 5A), while with the inducible system, PV expression levels decreased to 45% after 4 days of IPTG treatment and to less than 40% after 7 days of IPTG treatment (Fig 5B). IHC for PV using a fluorescent secondary antibody confirmed the strong silencing effect of the constitutive lentiviral-mediated shRNA (Fig 5C). As the result of the PV-down-regulation was more robust with the constitutive shRNA expression system, this one was chosen for the determination of COX1 and ATP synthase subunit β levels by Western blot analyses. Lentiviral shRNA-silencing of ectopically expressed PV in MDCK cells led to a significant increase in COX1 compared to the untreated PV-positive cell clone PV15 (Fig 6). Also the mitochondrial volume determined by FACS analysis was increased after Pvalb mRNA silencing, from 65.8 ± 11.5% to 79.2 ± 14.1% (p<0.05). Thus, the changes induced by ectopic PV expression were reverted partially by Pvalb shRNA treatment documenting the bidirectional regulation in an identical setting. The partial reversal of the mitochondrial volume might be the result of the incomplete PV down-regulation by the shRNA approach. ATP synthase subunit β levels were unchanged after PV down-regulation (Fig 6), i.e. neither PV’s up- nor its subsequent down-regulation affects ATP synthase subunit β levels. These experiments clearly demonstrated that PV and COX1 were bi-directionally and inversely regulated in MDCK cells.

Bottom Line: With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated.Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected.In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

No MeSH data available.


Related in: MedlinePlus