Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine
Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL),...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Karger Publishers
2015-09-01
|
| Series: | Kidney & Blood Pressure Research |
| Subjects: | |
| Online Access: | http://www.karger.com/Article/FullText/368524 |
| _version_ | 1818419497186361344 |
|---|---|
| author | Ioana Alesutan Katharina Musculus Tatsiana Castor Kousi Alzoubi Jakob Voelkl Florian Lang |
| author_facet | Ioana Alesutan Katharina Musculus Tatsiana Castor Kousi Alzoubi Jakob Voelkl Florian Lang |
| author_sort | Ioana Alesutan |
| collection | DOAJ |
| description | Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH4Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH4Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H+ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H+ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments. |
| first_indexed | 2024-12-14T12:39:30Z |
| format | Article |
| id | doaj.art-16ddce98083f4308a5b5adbcdf7369d4 |
| institution | Directory Open Access Journal |
| issn | 1420-4096 1423-0143 |
| language | English |
| last_indexed | 2024-12-14T12:39:30Z |
| publishDate | 2015-09-01 |
| publisher | Karger Publishers |
| record_format | Article |
| series | Kidney & Blood Pressure Research |
| spelling | doaj.art-16ddce98083f4308a5b5adbcdf7369d42022-12-21T23:00:57ZengKarger PublishersKidney & Blood Pressure Research1420-40961423-01432015-09-0140549049910.1159/000368524368524Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and MethylamineIoana AlesutanKatharina MusculusTatsiana CastorKousi AlzoubiJakob VoelklFlorian LangBackground/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH4Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH4Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H+ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H+ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.http://www.karger.com/Article/FullText/368524PhosphateTGFß1NFAT5Vesicular pHH+ pumpAlkaline phosphataseOsteoinductive signalingCalcificationSenescence |
| spellingShingle | Ioana Alesutan Katharina Musculus Tatsiana Castor Kousi Alzoubi Jakob Voelkl Florian Lang Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine Kidney & Blood Pressure Research Phosphate TGFß1 NFAT5 Vesicular pH H+ pump Alkaline phosphatase Osteoinductive signaling Calcification Senescence |
| title | Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine |
| title_full | Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine |
| title_fullStr | Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine |
| title_full_unstemmed | Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine |
| title_short | Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine |
| title_sort | inhibition of phosphate induced vascular smooth muscle cell osteo chondrogenic signaling and calcification by bafilomycin a1 and methylamine |
| topic | Phosphate TGFß1 NFAT5 Vesicular pH H+ pump Alkaline phosphatase Osteoinductive signaling Calcification Senescence |
| url | http://www.karger.com/Article/FullText/368524 |
| work_keys_str_mv | AT ioanaalesutan inhibitionofphosphateinducedvascularsmoothmusclecellosteochondrogenicsignalingandcalcificationbybafilomycina1andmethylamine AT katharinamusculus inhibitionofphosphateinducedvascularsmoothmusclecellosteochondrogenicsignalingandcalcificationbybafilomycina1andmethylamine AT tatsianacastor inhibitionofphosphateinducedvascularsmoothmusclecellosteochondrogenicsignalingandcalcificationbybafilomycina1andmethylamine AT kousialzoubi inhibitionofphosphateinducedvascularsmoothmusclecellosteochondrogenicsignalingandcalcificationbybafilomycina1andmethylamine AT jakobvoelkl inhibitionofphosphateinducedvascularsmoothmusclecellosteochondrogenicsignalingandcalcificationbybafilomycina1andmethylamine AT florianlang inhibitionofphosphateinducedvascularsmoothmusclecellosteochondrogenicsignalingandcalcificationbybafilomycina1andmethylamine |