Integrin engagement-induced inhibition of human myelopoiesis is mediated by proline-rich tyrosine kinase 2 gene products.
OBJECTIVE
Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells.


METHODS
Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differentiation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays.


RESULTS
CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors.


CONCLUSIONS
These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis.
Dylla
Scott J
SJ
Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, Minn, USA.
Deyle
David R
DR
Theunissen
Koen
K
Padurean
Adrian M
AM
Verfaillie
Catherine M
CM
D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003114:Colony-Forming Units Assay; D003470:Culture Media; D005353:Fibronectins; D051418:Focal Adhesion Kinase 2; D018922:HL-60 Cells; D006801:Humans; D016023:Integrins / Q000502:physiology*; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000473:pathology*; D038042:Myelopoiesis / Q000187:drug effects / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012710:Serum Albumin, Bovine; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology
D003470:Culture Media; D005353:Fibronectins; D016023:Integrins; D007527:Isoenzymes; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D012710:Serum Albumin, Bovine; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2