la vitamina k che ruolo ha?
DESCRIPTION
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.TRANSCRIPT
Turin, June 21-24, 2011
IN VITRO VITAMIN K TREATMENT IS NOT CAPABLE TO RESCUE UNEFFICIENT MGP-CARBOXYLATION IN PXE FIBROBLASTSAnnovi G., Boraldi F., Quaglino D.Dept. Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
Phylloquinone (vitamin K1)
Menaquinone (vitamin K2)
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
pseudoxanthoma elasticum
Ectopic calcification occurence:
1. Circulating nucleational complexes (high Ca / P)2. Cell death3. Alteration of NF-kB activity4. Proteolysis and ECM degradation5. Presence of “Bone Proteins”6. Loss of inhibitors
a 14-kDa secreted protein containing 5-glutamic acid residues that must be γ-carboxylated by a vitamin K-dependent γ-carboxylase in order to acquire calcium-binding properties.
Matrix Gla Protein (MGP)
-COOH
Vit. K1H2
CO2 + O2
Vit. K1
VK
OR
Vit. K1>O
Vitamin K CycleGamma-
carboxylase
MGP
MGP
GLA
GLA
GLA GLA GLA
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 Kng/ml
40 proteins differentiallyexpressed between control andPXE fibroblasts
CO2 + O2
Vit. K1H2
Vit. K1>O
Vitamin K cycle
Vit. K1
VK
OR
gamma-carboxylase
-COOHMGP
MGP
CALUCALU
e-
PDI
SH SH
PDI
S S
SHSHs s
50
37
0
40
80
120
160 *
CALU
PDI
75
50
C PXE
Control PXE K1 100µM K1 100µM K1 1µM K1 1µM K1 0.01µMK1 0.01µM0.00
0.25
0.50
0.75
1.00
1.25
1.50 Controllo PXE
*
*p<0.05 PXE vs ControlA
rbit
rary
un
it D
H2
Control PXE K2 100µM K2 100µM K2 1µM K2 1µM K2 0.01µMK2 0.01µM0.00
0.25
0.50
0.75
1.00
1.25
Control PXE
K2 100 µM K2 1 µM
* p<0.05 PXE vs Control$ p<0.05 Vitamin K2 PXE vs PXE
$ $
*
Arb
itra
ry u
nit
DH 2
Control PXE K1 100µM K1 100µM K1 1µM K1 1µM K1 0.01µMK1 0.01µM0.0
0.5
1.0
1.5
2.0
Control PXE
Arb
itra
ry u
nit
H2O
2
Control PXE K2 100µM K2 100µM K2 1µM K2 1µM K2 0.01µMK2 0.01µM0.0
0.5
1.0
1.5
2.0
K2 100 µM K2 1 µM K2 0.01 µM
Control PXE
Arb
itra
ry u
nit
H2O
2
0.1µM 100µM
Vitamin K2
UT DMF UT DMF 0.1µM 100µM 0.1µM 100µM
Vitamin K1
0.1µM 100µM
1± 0
.15
0.97
± 0.
29
1.2
± 0.2
5
0.94
± 0.
1
1.06
± 0.
131.
34 ±
0.32
1.70
± 0.
51
0.99
± 0.
15
1.62
± 0.
33
1.10
± 0
.11
1.07
± 0.
491.
42 ±
0.27
Control Control ControlPXE PXEPXE
Control PXE Control PXE0.0
0.5
1.0
1.5
*
*
Rel
ativ
e fo
ld c
han
ges
PDI CALU
DMFDMFK1 0.01mMK1 0.01mMK1 100mMK1 100mM0,1 0,1 100 1000.0
0.5
1.0
1.5 Control PXE
Rel
ativ
e fo
ld c
han
ges
(CA
LU
)
100 µM 0.1 µMDMF 100 µM 0.1 µM
**
** *
** *
Vit K1 Vit K2
DMFDMFK1 0.01mMK1 0.01mMK1 100mMK1 100mMK2 0.01mMK2 0.01mMK2 100mMK2 100mM0.0
0.5
1.0
1.5
2.0
100µM 0.1µMDMF
ControlPXE
100µM 0.1µM
Rela
tive f
old
ch
an
ges (
PD
I)
Vit K1 Vit K2
DMF CDMF P0.1 K10.1K1100 K1100 K10.1 K20.1 K2100 K2 C100 K2 P0
1
2
3
100 µM 0.1 µMDMF 100 µM 0.1 µM
* *
ControlPXE
Rel
ativ
e m
RN
A f
old
ch
ang
e
Solv Solv0,1µM0,1µM100µM100µM0,1M0,1mM100M100M0.0
0.5
1.0
1.5 Control PXE
Rel
ativ
e fo
ld c
han
ges
(MG
P)
100 µM 0.1 µMDMF 100 µM 0.1 µM
*
*
*
Vit K1 Vit K2
0
1
2
3
100µM0,1µM 100µM0,1µM
Control PXE
Gla
-MG
P (
a.u
.)
Vitamin K1 Vitamin K2
Control PXE
100µ
M0.
1µM
Vitamin K2
100µ
M0.
1µM
Vitamin K1
Control PXE
0,1µM K2100µM K10,1µM K1 100µM K2DMF
Con
trol
PX
E
Days of culture DMF Vitamin K1 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
Day 20
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 #
Day 30
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)
# morphological alteration
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