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

  2. > 21 Connexin genes cloned What is a connexin? Connexin Hemichannels Gap junction channels

  3. Connexin-43 in hematopoiesis • 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).

  4. 3a 1 2 3b Putative channel microanatomy in the BM stem cell niche Connexin-43 Small-molecule receptors

  5. How to dissect the specific contribution of Cx43 to hematopoiesis? Mice Vav1-Cre;Cx43flox/flox HSC WT KO Mice Col1a-Cre;Cx43flox/flox CFU-F WT1 WT2 KO1 KO2 Cx43 b-actin

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

  7. LT-HSC frequency population in BM is reduced in HSC/P Cx43-deficient mice LT-HSC * Percentage of Lin-/IL7Ra- Cx43-WTCx43-KO ST-HSC Percentage of Lin-/IL7Ra- Cx43-WTCx43-KO * p<0.05

  8. Deletion of Cx43 in HSC/P decreases the cycling fraction of LT-HSC WT Cx43KO LT-HSC ST-HSC * G0 G1 S G2/M G0 G1 S G2/M * p<0.05

  9. ** ANC (x103/mm3) Days after 5-FU treatment WT KO Deficiency of Cx43 in HSC/P impairs the hematopoietic response after 5-FU administration 50 10000 40 ** 8000 ** 30 ** 6000 Platelets (x103/mm3) 20 4000 10 2000 0 0 0 5 10 15 0 5 10 15 Days after 5-FU treatment ** p<0.01

  10. Cycling LT-HSC population in Cx43-deficient HSC is decreased as early as 48 hours after 5-FU administration % S-phase in LSK34- population (48h after 5-FU) ** Cx43KO Cx43WT ** p<0.01

  11. Deletion of Cx43 in HSC/P up-regulates the CDKI p21cip1 in LT- and ST-HSC and decreases cyclin D1 expression in LT-HSC ST-HSC LT-HSC ** * ** Cyclin D1 P53 P21 Cyclin D1 P53 P21 WT Cx43KO **p<0.01

  12. Summary I • 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.

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

  14. Content of stromal progenitors is increased in Cx43-deficient BM stroma 30 ** 25 20 15 CFU-F per 5x105 BM cells 10 5 0 WTCx43 KO ** p<0.01

  15. Radioprotective effect of progenitor transplantation is severely reduced in stromal Cx43-deficient mice CD45.1 CD45.1+ WT WT Limiting dilution assay 104 BM cells 110 Cx43WT Cx43KO 100 90 11.75 Gy 80 70 ** 60 Cumulative survival (%) CD45.2 CD45.2+ 50 Cx43 WT 40 30 20 10 0 11.75 Gy CD45.2+ CD45.2 0 10 20 30 Time (days) Cx43 KO ** p<0.01

  16. Homing of progenitors is reduced in stromal Cx43-deficient mice 8 16 hours after transplant 6 Homing to BM (%, CFU-C) 4 * 2 0 Cx43-WTCx43-KO There was no homing defect of Cx43KO hematopoietic progenitors into WT microenvironment * p<0.05

  17. G-CSF-induced mobilization of progenitors is reduced in stromal Cx43-deficient mice 4000 ** 3000 CFU-C per ml of blood 2000 1000 0 WT KO WT KO Basal G-CSF ** p<0.01

  18. CXCL12 expression and secretion are increased in HM Cx43-deficient BM Cx43-WTCx43-KO 1 2 3 1 2 3 CXCL12 b-actin * Cx43-WTCx43-KO *p<0.05

  19. Summary II 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.

  20. 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.

  21. 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 Our laboratory: Jose A. Cancelas Gabriel Ghiaur Lina Li Amitava Sengupta Jorden Arnett Susan Dunn Nicole Worsham