Function of CDK9 in cardiac hypertrophy

14th June 2010

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

PBS PE

WT NDPEPBS

H&E

Masson’sTrichrome

Markers of cardiac hypertrophy

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