Connexin-43 regulates the cell cycle entry of HSC and the migration of progenitors towards and from BM Daniel Gonzalez-Nieto, PhD

Hemichannels Gap 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;Cx43 flox/flox WT KO HSC Mice Col1  -Cre;Cx43 flox/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 in HSC/P Cx43-deficient mice Percentage of Lin - /IL7R  - * LT-HSC ST-HSC Percentage of Lin - /IL7R  - * p<0.05 Cx43-WT Cx43-KO

* G 0 G 1 S G 2 /M Deletion of Cx43 in HSC/P decreases the cycling fraction of LT-HSC WT Cx43KO LT-HSCST-HSC * p<0.05

Platelets (x10 3 /mm 3 ) Days after 5-FU treatment ** Days after 5-FU treatment ANC (x10 3 /mm 3 ) WT KO ** Deficiency of Cx43 in HSC/P impairs the hematopoietic response after 5-FU administration ** 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-phase in LSK34- population (48h after 5-FU) Cx43WT Cx43KO

WT Cx43KO Cyclin D1 P53 P21 ** ** p<0.01 Cyclin D1 P53 P21 Deletion of Cx43 in HSC/P up-regulates the CDKI p21 cip1 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 G o 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. p21 cip1 expression is up-regulated in Cx43-KO HSC in parallel with the increment of G o 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 CFU-F per 5x10 5 BM cells

** p<0.01 Radioprotective effect of progenitor transplantation is severely reduced in stromal Cx43-deficient mice ** 10 4 BM cells Cx43WT Cx43KO CD45.1 WT CD45.2CD45.2+ CD45.1+ WT CD45.2CD Gy Limiting dilution assay Cx43 WT Cx43 KO Time (days) Cumulative survival (%)

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 Homing to BM (%, CFU-C)

G-CSF-induced mobilization of progenitors is reduced in stromal Cx43-deficient mice ** G-CSFBasal WT KO CFU-C per ml of blood ** p<0.01

 -actin Cx43-WT Cx43-KO CXCL12 CXCL12 expression and secretion are increased in HM Cx43-deficient BM Cx43-WT Cx43-KO * 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. Cancelas Gabriel Ghiaur Lina Li Amitava Sengupta Jorden Arnett Susan Dunn Nicole Worsham Acknowledgements New York University School of Medicine Glenn Fishman David E Gutstein Washington University School of Medicine Roberto Civitelli Hospital Ramon & Cajal, Madrid Luis C. Barrio