La vitamina K che ruolo ha?
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La vitamina K è in grado di alterare l'espressione di proteine coinvolte nei processi di calcificazione? Per rispondere a questa domanda sono state fatte prove su cellule umane e su topi. Le analisi hanno riguardato l'espressione dei geni, delle proteine e delle modificazioni post-traduzionali a cui possono andare incontro.
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La vitamina K che ruolo ha?
- 1. IN VITRO VITAMIN K TREATMENT IS NOT CAPABLE TO RESCUE UNEFFICIENT MGP-CARBOXYLATION IN PXE FIBROBLASTS Annovi G., Boraldi F., Quaglino D. Dept. Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Turin, June 21-24, 2011
- 2. Phylloquinone (vitamin K1) Menaquinone (vitamin K2)
- 3. The inappropriate biomineralization occurring in soft tissue is defined as ectopic calcification • • • • • atherosclerosis chronic renal disease diabetes treatment with anti-coagulant therapy specific gene defects
- 5. Ectopic calcification occurence: 1. Circulating nucleational complexes (high Ca / P) 2. Cell death 3. Alteration of NF-kB activity 4. Proteolysis and ECM degradation 5. Presence of “Bone Proteins” 6. Loss of inhibitors
- 6. Matrix Gla Protein (MGP) GLA GLA GLA GLA GLA a 14-kDa secreted protein containing 5-glutamic acid residues that must be γcarboxylated by a vitamin Kdependent γ-carboxylase in order to acquire calciumbinding properties. Vit. K1H2 VKOR MGP Vit. K1 CO2 + O2 Vitamin K Cycle Gammacarboxylase Vit. K1>O MGP -COOH
- 7. Lab Invest. 2010 Jun;90(6):895-905. Epub 2010 Apr 5. Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome. Vanakker OM, Martin L, Schurgers LJ, Quaglino D, Costrop L, Vermeer C, Pasquali-Ronchetti I, Coucke PJ, De Paepe A. Serum Gla MGP % Serum Vit K ng/ml
- 9. 40 proteins differentially expressed between control and PXE fibroblasts
- 10. s s SH SH Vit. K1H2 e- MGP PDI S S VKOR CO2 + O2 PDI Vitamin K cycle gamma-carboxylase SH SH CALU CALU Vit. K1>O Vit. K1 75 50 PDI 50 37 * 160 80 120 CALU 40 0 C PXE MGP -COOH
- 12. 1.50 Controllo Control Arbitrary unit DH 2 Arbitrary unit DH 2 1.25 PXE PXE * * 1.25 1.00 1.00 $ $ 0.75 0.75 0.50 0.50 0.25 0.25 0.00 Control Control PXE PXE K2 100 µM K2 µM µM K2 1K2 1 µM1 µM K2 0.01 µMK2 0.01 µM K1 100 µM K1 100 µM K1 1µM K1 µM K2 1µM K1 0.01 µMK1 0.01 µM K2 100 100 * p<0.05 PXE vsControl *p<0.05 PXE vs Control $ p<0.05 Vitamin K2 PXE vs PXE Control PXE Control Arbitrary unit H2O2 Arbitrary unit H2O2 1.5 1.0 0.5 0.0 PXE 2.0 2.0 Control PXE K1 100 µM K1 100 µM K1 1 µM K1 1 µM K1 0.01 µMK1 0.01 µM 1.5 1.0 0.5 0.0 Control PXE K2 100 µM K2 100 µM K2 1K2 1 µM 1 µM K2 0.01 0.01 µM µM K2 µM K2 100 µM K2 µMK2 0.01
- 13. UT DMF 0.1µM 100µM 0.1µM 100µM Vitamin K1 Control PXE Control 0.1µM 100µM 1.7 0 ± 0.5 1 0.1 5 ± 0.9 9 1.6 2 ± 0.3 3 0.1 1 ± 1.1 0 0.2 7 1.4 2± 0.4 9 1.0 7 ± 0.3 2 1.3 4 ± 0.1 3 ± 1.0 6 0.1 0.9 4 ± 0.2 5 ± 1.2 0.2 9 ± 0.9 7 1± 0.1 5 UT DMF 0.1µM 100µM Vitamin K2 PXE Control PXE
- 14. Relative fold changes 1.5 * 1.0 * 0.5 0.0 Control PXE 2.0 Control PXE 1.5 1.0 0.5 0.0 DMFDMFK1 0.01mM 100mM 0.1µM K2 100mM K1 0.01mM K2 0.01mM K2 K2 100µM DMF 0.1µM K1 K1 100mM 0.01mM 100mM 100µM Vit K1 Vit K2 PXE CALU Relative fold changes (CALU) Relative fold changes (PDI) PDI Control 1.5 Control PXE 1.0 * 0.5 0.0 * * * * * * * DMFDMFK1 0.01mM 100mM0.1 µM 100 µM 100 µM DMF 0.1 K1 0.01mM 100mM µM K1 K1 0,1 0,1 100 100 Vit K1 Vit K2
- 15. Relative mRNA fold change 3 2 1 * 0 1.5 Relative fold changes (MGP) Control PXE * DMFDMF0.1 K1 100 K1 K1 K2 K2 K2 CK2 P C 0.1 µM 100 µM 0.1 0.1 100 µM DMF P 0.1K1 100 0.1 µM 100 100 Control PXE 1.0 0.5 * * * 0.0 Solv Solv0,1µM µ M 100µM 0,1 100µM 0,1M0,1mM 100M 100M 100 µM DMF 0.1 µM Vit K1 0.1 µM 100 µM Vit K2
- 16. Control PXE 2 1 0 0,1µM 0,1µM 100µM Vitamin K1 100µM Vitamin K2 Control PXE PXE 0.1µM 0.1µM Control 100µM 100µM Gla-MGP (a.u.) 3 Vitamin K1 Vitamin K2
- 17. Control PXE DMF Days of culture 100µM K1 0,1µM K1 DMF 0,1µM K2 Vitamin K1 100µM K2 Vitamin K2 0.1µM 100µM 0.1µM 100µM Day 10 Control 0.2 ±0.20 0.4 ±0.25 0.6 ±0.40 0.7 ±0.66 0.8 ±0.48 PXE 0.2 ±0.17 0.4 ±0.40 0.8 ±0.58 0.3 ±0.28 0.4 ±0.24 Control 0.3 ±0.25 1.4 ±0.87 2.6 ±1.39 0.7 ±0.67 2.0 ±0.81 # PXE 1.2 ±0.56 1.8 ±1.11 4.6 ±2.64 3.4 ±2.57 1.7 ±1.66 # Control 9.9±3.28 9.4 ±5.01 9.0 ±5.81 26.0 ±7.72 n.d.b) PXE 14.4 ±4.15 12.0±4.3 11.9±3.86 22.2 ±5.76 n.d. b) Day 20 Day 30 # morphological alteration
- 18. In these work we have demontrated that: # PXE fibroblasts are able to respond to vitamin K increasing the efficiency of the carboxylation process. # vitamin K2 reduce the expression of MGP mRNA and protein. #vitamin k have negligible effects on MGP carboxylation #vitamin k are not able to counteract the in vitro mineralization process. ° mice data °in vivo data
Editor's Notes
- Vitamin K is a group of fat soluble vitamins that, in their reduced form, are needed for the posttranslational modification of a number of proteins involved in blood coagulation as well as in the mineralization process.
- Development of ectopic calcifications is responsible for clinical complications occurring during atherosclerosis, chronic renal diseases, diabetes and upon treatments with warfarin, or as the result of specific gene defects.
- Manca l'occhio pseudoxanthoma elasticum (PXE), a rare genetic disorder associated to mutations in the ABCC6 gene, and characterized by the progressive mineralization of elastic fibers (Le Saux 2000). Clinical manifestations mainly affect skin with laxity and formation of xantomatous papule; eyes, with angioid streaks and retinal haemorrhages leading to central vision loss; and the cardiovascular system with premature intermittent claudication and higher frequency of angina and myocardial infarction
- Ectopic calcification may occur (1) when the systemic concentrations of calcium and phosphate in extracellular fluid exceed the saturation point (metastatic calcification); (2) as a consequence of the replacement or transition of injured, degenerated, and necrotic tissue by mineral depositions (dystrophic calcification); or (3) by means of the transdifferentiation of mesenchymal cells into bone tissue (ectopic calcification) (
- Matrix Gla Protein (MGP) is an important local inhibitor of cartilage, arterial and soft tissues calcification (Ketteler M. et al., 2005). Several in vitro and in vivo studies have shown that MGP is expressed by many peripheral cell types, including fibroblasts and smooth muscle cells (Fraser J.D. and Price P.A., 1988; Shanahan C.M. et al., 1993). MGP is a small ECM protein that contains nine γ- carboxyglutamic acid residues, five of which are posttranslationally modified, resulting from vitamin K-dependent carboxylation of the protein in the endoplasmic reticulum (Price P.A. et al., 1983; Price P.A. and Williamson M.K., 1985). MGP γ- carboxylation is performed by the vitamin K-dependent enzyme, γ-carboxylase and this modification appears to be important in its function: conditions causing a relative deficiency of functional vitamin K may increase vascular calcification because of incomplete γ-carboxylation and reduced function of MGP (Jie K.S. et al., 1995)The case of mutations in the MGP gene that, due to the production of nonfunctional or absent MGP, is associated to abnormal cartilage and arterial mineralization, . Moreover, lack of function due to insufficient γ-carboxylation or to reduced levels of vitamin K rather than the amount of MGP, is the factor that increases the risk of calcification and is inversely correlated with the severity of coronary artery calcification (Jono S. et al., 2004).
- Reduced levels of vitamin K and the consequent reduction of carboxylated-Matrix Gla Protein (Gla-MGP) have been demonstrated by Vanakker and coworker in serum of pseudoxanthoma elasticum (PXE) patients
- In this paper we have demonstrated that PXE fibroblasts exhibited an insufficient vitamin-K-dependent carboxylation of MGP
- Moreover, we have recently reported that fibroblasts, isolated from PXE patients and cultured in vitro, have an altered protein profile
- And in particular, PXE firolasts have dispalyed a down-regulation of protein disulfide isomerase (PDI) and an up- regulation of calumenin (CALU), two proteins of the endoplasmic reticulum that are involved in vitamin K cycle, by assuring the appropriate reduced environment, necessary for regenerating the vitamin in its active form, and by inhibiting the γ-carboxylase activity, respectively Taken together these evidences, it could be conceivable that vitamin K supplementation might be capable to counteract the abnormal expression of proteins involved in vitamin K cycle and in MGP carboxylation in PXE fibroblasts. .