ACTIVATION OF THE SODIUM HYDROGEN EXCHANGER VIA THE FIBRONECTIN-INTEGRIN PATHWAY RESULTS IN HEMATOPOIETIC STIMULATION/

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellu lar microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulatio n of the intracellular acid-base conditions. One of the mechanisms by which intracellular pH (pH(i)) is regulated is through the sodium/hydr ogen exchanger, a ubiquitous membrane protein which exploits the intra - and extracellular sodium ion gradient to drive hydrogen ions out of the cell. However, activation of the exchanger via mitogenic and nonmi togenic signals leads to an increase in pH(i) which, in turn, may dire ctly or indirectly result in a proliferative response. It has been sho wn that interaction of fibronectin with its integrin receptor subunits alpha(4) and alpha(5) can result in activation of the Na+/H+ exchange r. In this report, we demonstrate that when mouse bone marrow cells ar e physically brought together in a preculture system we designate as h igh cell density culture (HCDC), in a small volume and at the same cel lularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the pres ence of growth factors. Neutralizing antibodies to the growth factors added to HCDC had little, if any, effect on the degree of stimulation. However, when antibodies to fibronectin or the a, integrin subunit we re added to HCDC, inhibition was observed, indicating that the observe d hematopoietic stimulation occurred via the fibronectin-integrin path way. Addition of 5 mu M 5-(N,N-hexamethylene) amiloride (5-HMA), a spe cific inhibitor of the Na+/H+ exchanger, also resulted in inhibition o f in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pH(i) of normal and HCDC-treated bone marrow cells in the absence and presence of 5-HMA by flow cytometry using the fluorescent pH-sensitive indicator, carboxy SNARF-1 AM. it was found that cells s ubjected to HCDC exhibited a higher pH(i) than normal fresh cells. In each case, the pH(i) was lowered in the presence of 5-HMA. Furthermore , addition of antibodies to fibronectin or the alpha(4) integrin subun it to HCDC also reduced the pH(i) to a similar level to that found for 5-HMA. Our results demonstrate, for the first time, that a hematopoie tic stem and progenitor cell proliferative response can be initiated b y activation of the Na+/H+ exchanger, leading to an increase in pH(i), via cell-cell interaction through the fibronectin-integrin pathway. T his pathway could, therefore, be significant not only in normal hemato poietic regulation, but also under pathophysiological conditions, J. C ell. Physiol. 177:109-122, 1998. (C) 1998 Wiley-Liss, Inc.