CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions

Abstract

The chemokine CXCL12 is essential for the function of hematopoietic stem and progenitor cells. Here we report that secretion of functional CXCL12 from human bone marrow stromal cells (BMSCs) was a cell contact–dependent event mediated by connexin-43 (Cx43) and Cx45 gap junctions. Inhibition of connexin gap junctions impaired the secretion of CXCL12 and homing of leukocytes to mouse bone marrow. Purified human CD34+ progenitor cells did not adhere to noncontacting BMSCs, which led to a much smaller pool of immature cells. Calcium conduction activated signaling by cAMP–protein kinase A (PKA) and induced CXCL12 secretion mediated by the GTPase RalA. Cx43 and Cx45 additionally controlled Cxcl12 transcription by regulating the nuclear localization of the transcription factor Sp1. We suggest that BMSCs form a dynamic syncytium via connexin gap junctions that regulates CXC12 secretion and the homeostasis of hematopoietic stem cells.

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Figure 1: Secretion of functional CXCL12 in the bone marrow correlates with expression of Cx43 and Cx45 and gap-junction activity.
Figure 2: CXCL12 secretion is cell contact dependent, but CXCL12 production is not.
Figure 3: Cx43 and Cx45 gap junctions directly regulate CXCL12 secretion.
Figure 4: Cx43 and Cx45 regulate Cxcl12 transcription by localizing Sp1 to the nucleus.
Figure 5: Calcium internalization controls the secretion of CXCL12 from BMSCs.
Figure 6: Calcium internalization results in more cAMP and PKA activation, which in turn induces the secretion of CXCL12 from BMSCs via RalA.
Figure 7: Maintenance of CD34+CD38 primitive properties by stromal networks.

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Acknowledgements

We thank J. Von Maltzen and K. Willecke (Institute of Genetics Division of Molecular Genetics, University of Bonn, Bonn, Germany) for BMSCs with loxP-flanked Cx45 that express Cre from the myxovirus promoter; G. Lalli (King's College London) for plasmids encoding Myc tag alone or Myc-tagged dominant negative RalA; M. Segal for helping with real-time calcium imaging; R. Alon, N. Dekel and I.H. Oh for discussions; and C.M. Sorensen for advice on connexin mimetic peptides. Supported by the Israeli Science Foundation (ISF 544/09), the European Union (Advance Cell-based Therapies for the Treatment of Primary Immunodeficiency HEALTH-F5-2010-261387) and The Edith Arnoff Stein Professorial Chair in Stem Cell Research (T.L.).

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A.S. designed and did the research, collected and analyzed data, and wrote the manuscript; T.I., G.D., A.K., K.G. and A.L. helped with designing and doing experiments; D.C. did and analyzed real time calcium imaging; Z.S. did dominant negative experiments; A.A. and A.N. supplied human bone marrow aspiration samples and mobilized blood samples from healthy donors; O.K. helped with designing experiments; R.S. designed the research; and T.L. designed the research and wrote the manuscript.

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Correspondence to Tsvee Lapidot.

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The authors declare no competing financial interests.

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Schajnovitz, A., Itkin, T., D'Uva, G. et al. CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions. Nat Immunol 12, 391–398 (2011). https://doi.org/10.1038/ni.2017

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