Connexin-43 regulates the cell cycle entry of HSC and the migration of progenitors

towards and from BM

Daniel Gonzalez-Nieto, PhD

HemichannelsGap junction

channels

What is a connexin?

> 21 Connexin genes cloned

Connexin

• HSC function requires cell contact with pre-osteoblastic stromal cells (Xie Y et al, Nature 2009).

• Cx43 is expressed by adult BM osteoblasts & stromal cells (Lecanda F et al., MBC 2003; Cancelas J.A., et al., Blood 2000).

• Cx43 is indispensable for osteoblast function (Lecanda F., J. Cell Biol, 2000).

• Cx43 is expressed by HSC and downregulated during differentiation to MPP and differentiated cells (Forsberg C et al., PLoS Genetics, 2005; Chambers SM et al., Cell Stem Cell, 2007).

• Cx43 fetal liver hematopoiesis is impaired in Cx43-deficiency mice (Cancelas J.A., et al., Blood 2000).

• 5-FU induces overexpression of BM Cx43 in vivo (Rosendaal M et al., J. Cell. Sci, 1994).

Connexin-43 in hematopoiesis

1

Connexin-43

Small-molecule receptors

2

3a

3b

Putative channel microanatomy in the

BM stem cell niche

How to dissect the specific contribution of Cx43 to hematopoiesis?

Mice Vav1-Cre;Cx43flox/flox

Rel

ativ

e m

RN

A

Cx4

3 ex

pre

ssio

n0

20

40

60

80

100

120

WT KO

HSC

Mice Col1-Cre;Cx43flox/flox

Cx43

WT1 WT2 KO1 KO2

-actin

CFU-F

What is the phenotype of Cx43-deficient HSC/P?

LT-HSC frequency population in BM is reduced inHSC/P Cx43-deficient mice

Pe

rce

nta

ge

of

Lin

- /IL

7R

-

*

LT-HSC

ST-HSC

Pe

rce

nta

ge

of

Lin

- /IL

7R

-

* p<0.05

Cx43-WT Cx43-KO

Cx43-WT Cx43-KO

% c

ell c

ycle

0

20

40

60

80

100 *

% c

ell c

ycle

0

20

40

60

80

100

G0 G1 S G2/M G0 G1 S G2/M

Deletion of Cx43 in HSC/P decreases the cycling fraction of LT-HSC

WT

Cx43KO

LT-HSC ST-HSC

* p<0.05

Pla

tele

ts(x

103 /

mm

3 )Days after 5-FU treatment

**

**

Days after 5-FU treatment

AN

C(x

103 /

mm

3 )

WTKO

**

**

Deficiency of Cx43 in HSC/P impairs the hematopoietic response after 5-FU administration

0 5 10 150

10

20

30

40

50

0 5 10 150

2000

4000

6000

8000

10000

** p<0.01

Cycling LT-HSC population in Cx43-deficient HSC is decreased as early as 48 hours after 5-FU administration

** p<0.01

**

% S

-pha

se in

LS

K34

- po

pula

tion

(48h

aft

er 5

-FU

)

Cx43WT Cx43KO

Rel

ativ

e m

RN

A

exp

res

sio

n

0

2

4

6

8

WT

Cx43KO

Cyclin D1 P53 P21

**

** p<0.01

Cyclin D1 P53 P21

Deletion of Cx43 in HSC/P up-regulates the CDKI p21cip1 in LT- and ST-HSC

and decreases cyclin D1 expression in LT-HSC

**

*

LT-HSC ST-HSC

1. Mice with Cx43-deficit in the HSC/P show altered

hematopoiesis recovery response after 5-FU treatment.

2. The absence of Cx43 expression in LT-HSC induces an increment in the Go state, reducing the proliferative activity of this population, a phenomenon that could justify the delay in the hematopoiesis recovery after 5-FU.

3. Cx43-deficient LT-HSC show decreased proliferation as early as 48 hours after 5-FU administration.

4. p21cip1 expression is up-regulated in Cx43-KO HSC in parallel with the increment of Go state, suggesting that Cx43 controls different pathways implicated in cell cycling.

Summary I

What is the BM phenotype of Cx43-deficiency in the stroma?

** p<0.01

WT Cx43 KO

**

Content of stromal progenitors is increased in Cx43-deficient BM stroma

CF

U-F

per

5x

105 B

M c

ells

0

5

10

15

20

25

30

** p<0.01

Radioprotective effect of progenitor transplantation isseverely reduced in stromal Cx43-deficient mice

**

104 BM cells

Cx43WTCx43KO

CD45.1

WT

CD45.2CD45.2+

CD45.1+

WT

CD45.2CD45.2+

11.75 Gy

11.75 Gy

Limiting dilution assay

Cx43WT

Cx43KO

Time (days)0 10 20 30C

um

ula

tive

su

rviv

al (

%)

0102030405060708090

100110

Cx43-WT Cx43-KO

*

* p<0.05

Homing of progenitors is reduced in stromal Cx43-deficient mice

16 hours after transplant

There was no homing defect of Cx43KO hematopoietic progenitors

into WT microenvironment

0

2

4

6

8

Ho

min

g t

o B

M (

%,

CF

U-C

)

G-CSF-induced mobilization of progenitors is reduced in stromal Cx43-deficient mice

**

G-CSFBasal

WT KO WT KO

CF

U-C

pe

r m

l of

blo

od

0

1000

2000

3000

4000

** p<0.01

-actin

1 2 3 1 2 3

Cx43-WT Cx43-KO

CXCL12

CXCL12 expression and secretion are increased in HM Cx43-deficient BM

Cx43-WT Cx43-KO

BM

CX

CL

12 s

ecre

tio

n

(ng

/tw

o f

emu

rs)

0.0

0.5

1.0

1.5

2.0

* p<0.05

*

1. Radioprotective effect of progenitor transplantation is reduced in HM Cx43-

deficient mice and correlates with a specific progenitor homing defect.

2. G-CSF-induced progenitor mobilization is reduced in stromal Cx43-deficient

mice.

3. Expression and secretion of CXCL12 are increased in stromal Cx43-

deficient mice, correlating with an increased number of stromal

progenitors.

Summary II

Implications

Deficiency of Cx43 in BM induces, at least, two distinct phenotypes of the

HSC/P niche(s):

A. Deficiency of Cx43 in HSC impairs cell cycle entry which correlates with

decreased cyclin D1 and increased p21cip1 expression

B. Deficiency of Cx43 in the BM stroma induces expansion of

mesenchymal progenitors while impairs homing and mobilization

response to G-CSF.

Cx43 plays pleiotropic roles in the HSC niche, controlling HSC fitness, pool

size and movement within the marrow.

Our laboratory:

Jose A. CancelasGabriel GhiaurLina LiAmitava SenguptaJorden ArnettSusan DunnNicole Worsham

Acknowledgements

New York University School of Medicine

Glenn FishmanDavid E Gutstein

Washington University School of Medicine

Roberto Civitelli

Hospital Ramon & Cajal, Madrid

Luis C. Barrio