Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation.

Abstract

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellular microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulation of the intracellular acid-base conditions. One of the mechanisms by which intracellular pH (pHi) is regulated is through the sodium/hydrogen 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 nonmitogenic signals leads to an increase in pHi which, in turn, may directly or indirectly result in a proliferative response. It has been shown that interaction of fibronectin with its integrin receptor subunits alpha4 and alpha5 can result in activation of the Na+/H+ exchanger. In this report, we demonstrate that when mouse bone marrow cells are physically brought together in a preculture system we designate as high cell density culture (HCDC), in a small volume and at the same cellularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the presence 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 alpha4 integrin subunit were added to HCDC, inhibition was observed, indicating that the observed hematopoietic stimulation occurred via the fibronectin-integrin pathway. Addition of 5 microM 5-(N,N-hexamethylene) amiloride (5-HMA), a specific inhibitor of the Na+/H+ exchanger, also resulted in inhibition of in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pHi 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 subjected to HCDC exhibited a higher pH than normal fresh cells. In each case, the pH was lowered in the presence of 5-HMA. Furthermore, addition of antibodies to fibronectin or the alpha4 integrin subunit to HCDC also reduced the pH, to a similar level to that found for 5-HMA. Our results demonstrate, for the first time, that a hematopoietic stem and progenitor cell proliferative response can be initiated by activation of the Na+/H+ exchanger, leading to an increase in pHi, via cell-cell interaction through the fibronectin-integrin pathway. This pathway could, therefore, be significant not only in normal hematopoietic regulation, but also under pathophysiological conditions.