function of cdk9 in cardiac hypertrophy 14 th june 2010
TRANSCRIPT
Cardiovascular diseases - Cardiac hypertrophy
Basics and SymptomsBasics and SymptomsCardiovascular disease is today the main cause of mortality worldwide, Cardiovascular disease is today the main cause of mortality worldwide, representing 30% of all deaths.representing 30% of all deaths.
Among them, heart failure represents one of the most frequent type with Among them, heart failure represents one of the most frequent type with broad range of clinical symptoms and initial causes:broad range of clinical symptoms and initial causes:
- myocardial infarction- myocardial infarction- hypertension- hypertension- coronary artery diseases- coronary artery diseases- valvular diseases- valvular diseases- infectious diseases- infectious diseases- cardiotoxic substances- cardiotoxic substances- cardiac hypertrophy- cardiac hypertrophy
Cardiac hypertrophyCardiac hypertrophy is primarily characterized by Cardiac hypertrophy is primarily characterized by de-novode-novo growth of growth of differentiated cardiac myocytes, typically leading to, but not always, the differentiated cardiac myocytes, typically leading to, but not always, the enlargement of the entire myocardium.enlargement of the entire myocardium.
Heineke et al. Nature Reviews Molecular Cell Biology 7, 589–600 (August 2006) | doi:10.1038/nrm1983
Cardiac hypertrophy
AdaptiveAdaptive
PathologicalPathological
-Chronic exerciseChronic exercise
- Pregnancy- Pregnancy
Persistent Persistent hemodynamichemodynamicdemanddemand
Heart developmentHeart development - Fetal Gene Program- Fetal Gene Program
Molecular mechanismMolecular mechanismss of cardiac hypertrophy of cardiac hypertrophy
Cardiac myocytes could be attributed to overall increase of protein content due Cardiac myocytes could be attributed to overall increase of protein content due to elevated RNA production. Therefore it is no surprise that RNA polymerase II to elevated RNA production. Therefore it is no surprise that RNA polymerase II (RNAPII), responsible for transcription of coding RNA species, was identified (RNAPII), responsible for transcription of coding RNA species, was identified as a limiting factor of hypertrophic growth. as a limiting factor of hypertrophic growth.
http://ghr.nlm.nih.gov/handbook/illustrations/http://ghr.nlm.nih.gov/handbook/illustrations/proteinsyn.jpgproteinsyn.jpg
http://kvhs.nbed.nb.ca/gallant/biology/transcription.jpghttp://kvhs.nbed.nb.ca/gallant/biology/transcription.jpg
Function of P-TEFb in the regulation of transcription
P-TEFb – Positive Transcription Elongation Factor b
Peterlin BM, Price DH., 2006, “Controlling the elongation phase of transcription with P-TEFb”, Mol Cell. Aug 4;23(3):297-305.
7SK snRNA and P-TEFb
The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcriptionYang Z, Zhu Q, Luo K, Zhou Q., Nature. 2001 Nov 15;414(6861):317-22
7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexesNguyen VT, Kiss T, Michels AA, Bensaude O., Nature. 2001 Nov 15;414(6861):322-5
7SK snRNA7SK snRNA
pppGpppG
UUUU-OHUUUU-OH
Cdk9Cdk9
CycCyc
Hexamethylene Bisacetamide inducible protein1- Hexim1
Cdk9Cdk9
CycCycHex1
Hex1
**CycCyc
Cdk9Cdk9
7SK snRNA7SK snRNA
There is also Hexim2 protein with function similar to Hexim1.There is also Hexim2 protein with function similar to Hexim1.
MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent mannerMichels et al., Mol Cell Biol. 2003 Jul;23(14):4859-69
Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNAYik JH et al., Mol Cell. 2003 Oct;12(4):971-82
MEPCE a LARP7
Cdk9Cdk9
CycCycHex1
MEPCEMEPCE
Hex1
**CycCyc
Cdk9Cdk9LARP7LARP7
A La-related protein modulates 7SK snRNP integrity to suppress P-TEFb-dependent transcriptional elongation and tumorigenesis.He N, Jahchan NS, Hong E, Li Q, Bayfield MA, Maraia RJ, Luo K, Zhou Q., Mol Cell. 2008 Mar 14;29(5):588-99. Epub 2008 Jan 31.
Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH, Coulombe B., Mol Cell. 2007 Jul 20;27(2):262-74.
Composition of P-TEFb
There are two major pools of P-TEFb in cells:
Small – active form of P-TEFb
Large – inactive form of P-TEFb
Cdk9Cdk9
CycCyc
LARGELARGEinactiveinactive
Cdk9Cdk9
CycCycHex1
MEPCEMEPCE
Hex1
**CycCyc
Cdk9Cdk9LARP7LARP7
SMALLSMALLactiveactive
UVUVStressStress
CytokinesCytokinesInhibitorsInhibitors
Ectopic expression of CycT1 leads tocardiac hypertrophy in mice
Sano et al., 2002, Nat Med, 1310-1317
Function of P-TEFb in cardiac hypertrophy
Kulkarni et al., 200 et al., Recent Prog Horm Res. 59:125-39
Cdk9Cdk9CycCyc
Hex1
MEPCEMEPCE
Hex1
**CycCyc
Cdk9Cdk9LARP7LARP7
Cdk9Cdk9
CycCyc
He Br Lu Sm Sp Ki Th Li Te Pa
cycT1
-/- (ND)+/+
He Br Lu Sm Sp Ki Th Li Te Pa
*
Protein levels of P-TEFb subunits in mouse organs
CDK9-55
CDK9-42
Hex1
Severity of cardiac hypertrophy in WT and ND CycT1 mice
HW/TL – heart weigth / tibia length, PE – Phenylephrine, PBS – Phosphate Saline Buffer
t-Test: Two-Sample Assuming Unequal Variances
MeanVarianceObservationsHypothesized Mean Differencedft StatP(T<=t) one-tailt Critical one-tailP(T<=t) two-tailt Critical two-tail
Cyclin T1 KO Hypertrophy
0
1
2
3
4
5
6
7
8
WT ND
HW
/BW PE
PBS
Post-translational modification of CDK9and CTD phoshorylation
CDK9CDK9
WT KO
CCycT1ycT1
PE
PE
PB
SP
BS
PE
PE
PB
SP
BS
PE
PE
PE
PE
PE
PE
PB
SP
BS
PB
SP
BS
PE
PE
PE
PE
PB
SP
BS
PE
PE
PE
PE
PB
SP
BS
TH
-WT
TH
-WT
TH
-ND
TH
-ND
TH
-WT
TH
-WT
TH
-ND
TH
-ND
PB
SP
BS
PE
PE
Ser-2
Ser-5 P
P
KO
Phosphatase effect on post-translational modification of 42-CDK9 form
Inpu
tIn
put
++ ---PhosphatasePhosphatase
IP-CDK9IP-CDK9
CDK9CDK9
CDK9CDK9
CIPCIP ++ --
InputInput
HEHE -/- -/-
HE
HE
WT
WT
HE
HE
DN
DN
Composition of P-TEFb complexes during cardiac hypertrophy in WT and ND CycT1 mice
PB
SP
BS
PE
PE
PE
PE
PB
SP
BS
WB: WB: CDK9CDK9
IP-IP-CCycT2ycT2IP-IP-CDK9CDK9
IP-IP-CCycT1ycT1
IP-IP-CCycT2ycT2
PB
SP
BS
PE
PE
PE
PE
PB
SP
BS
InputInput
WB: Hexim1WB: Hexim1
Input
WT KO WT KO
CycT2CDK9
Hexim1
CycT2CDK9
WT ND
inactiveinactive activeactive
I
II
IIIIV
I
IIIII
IVV
VI
I. MetabolismII. TGFIII. AutophagyIV. Wnt pathwayV. Cytokines/receptorsVI. Notch
I. MetabolismII. Cell communicationIII. Cell cycleIV. Cytokines/receptor
Groups of genes down-regulated after siRNA against CycT1 and CycT2
siRNA CycT1 siRNA CycT2
He at al., 2008, Mol Cel, 29, 588-599He at al., 2008, Mol Cel, 29, 588-599
Function of P-TEFb in breast cancer
He at al., 2008, Mol Cel, 29, 588-599He at al., 2008, Mol Cel, 29, 588-599
Function of P-TEFb in breast cancer
Function of Function of P-TEFb P-TEFb inin Leukemia Leukemia
Disease is caused by chromosomal abberations, most often translocations, Disease is caused by chromosomal abberations, most often translocations, affecting Chromosome 11 at band q23affecting Chromosome 11 at band q23Mostly chimeric proteins are generated between H3 Lisine 4-specific Mostly chimeric proteins are generated between H3 Lisine 4-specific methyltransferase (MLL) DNA-binding domain and new protein.methyltransferase (MLL) DNA-binding domain and new protein.This fusion proteins potently transform hematopoietic cells.This fusion proteins potently transform hematopoietic cells.
Mueller et al., PLoS Biol. 2009, Nov;7(11):e1000249. Epub 2009 Nov 24Mueller et al., PLoS Biol. 2009, Nov;7(11):e1000249. Epub 2009 Nov 24
MLL – Mixed-Lineage Leukemia is agressive subtype of acute leukemiaMLL – Mixed-Lineage Leukemia is agressive subtype of acute leukemia
Flovopiridol is tested in other leukemias: AML, ALL and CLLFlovopiridol is tested in other leukemias: AML, ALL and CLL
Small chemical inhibitors of P-TEFb
Kryštof et al., 2009, Med Res Rev, Sep 15
http://upload.wikimedia.org/wikipedia/commons/thumb/9/9e/Flavopiridol.svg/615px-Flavopiridol.svg.png
Flavopiridol
We are interested in
- Characterization of post-translational Characterization of post-translational modifications in CDK9modifications in CDK9
- Test of Flavopiridol and Olomoucine II in Test of Flavopiridol and Olomoucine II in management of cardiac hypertrophy management of cardiac hypertrophy symptoms in micesymptoms in mice
- Function of P-TEFb in cancer progressionFunction of P-TEFb in cancer progression
Acknowledgement:Acknowledgement:Matija B. PeterlinMatija B. PeterlinDalibor BlažekDalibor BlažekHuimin JiangHuimin JiangKolegům z laboratořeKolegům z laboratoře
Immunohistilogy core, Immunohistilogy core, Oracleman, VivianOracleman, Vivian
VláďVláďimimír Kryštofír KryštofMiroslav StrnadMiroslav Strnad