HAND proteins are tissue-restricted members of the basic helixloop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis.

Secondary amyloidosis is a systemic disease characterized by the extracellular tissue deposition of insoluble fibrillar amyloid A protein. Aberrant metabolism of serum amyloid A protein by reticuloendothelial cells is thought to result in the accumulation of fibrils within the tissue. Treatment of mice with amyloid-enhancing factor (AEF) in conjunction with an inflammatory stimulus (i.e., AgNO3) induced amyloid deposition within 48-72 h. The activation state of a macrophage largely defines its enzymatic capabilities. In the studies reported here, we examined the effect of AEF on spleen macrophage activation using both functional and phenotypic assays. We found that while AEF in the presence or absence of AgNO3 has no apparent effect on the ability of spleen and liver macrophages to phagocytose or kill Listeria monocytogenes, it appears to block enhanced respiratory burst function (as measured by O2- production) observed with AgNO3 alone. AEF therefore seems capable of inhibiting certain macrophage activation-associated functions while not affecting others. Our activation phenotype studies, using surface Ia expression, reveal that AEF blocks the increase in number of splenic macrophages expressing Ia seen with AgNO3 alone. Treatment with interferon-gamma was found to restore decreased Ia expression in animals given AEF+AgNO3 but did not prevent amyloid A fibril deposition.

The magnitude of the macrophage inflammatory response differs among inbred mouse strains. Mice of the A/J strain respond poorly to sterile inflammatory stimuli while those of the C57BL/6 strain show a strong response. Inflammatory macrophages found at the site of inflammation are the product of bone marrow (BM) myeloid stem cells. Mice of the A/J strain were found to have half the number of BM nucleated cells per femur than those of the C57BL/6 strain. The lower BM cellularity may be one reason for the poor macrophage inflammatory response observed in A/J mouse strain. Using A x B/B x A recombinant inbred mouse strains, we determined that the number of nucleated cells per femur found in normal mice was not a determining factor of the magnitude of the macrophage inflammatory response. One additional explanation for the poor macrophage inflammatory response in mice of the A/J strain is their deficiency in the C5 component of complement. Using a C5-sufficient A/J.C5 congenic strain, we have previously shown that the presence of C5 on the A/J background improved their inflammatory response. We compared A/J and A/J.C5 mouse strains to determine whether or not C5 had an impact on the BM cell response to inflammatory stimulus. The presence of C5 on the A/J background could contribute to the improvement of the inflammatory response in mice of the A/J.C5 strain by inducing a greater number of nucleated cells to exit the BM compartment early following induction of inflammation.

The multicolony stimulating factor Interleukin-3 (IL-3) has a role in regulating the proliferation, differentiation, and survival of myeloid stem cells and committed progenitor cells within each of the myeloid lineages. It has been referred to as an emergency factor appearing following triggering of an inflammatory response. The ability of bone marrow (BM) stem cells to respond to a stimulus such as IL-3 in vitro may reflect the in vivo capacity of BM stem cells to generate newly BM-derived macrophages being recruited to an inflammatory site. Both parameters, namely the BM cell response to in vitro IL-3 treatment and the magnitude of the macrophage inflammatory response vary among inbred mouse strains. Mice of the A/J strain are known to have weak macrophage inflammatory response to a phlogistic agent and their BM cells are hyporesponsive to IL-3 exposure. In contrast, mice of the C57BL/6 strain mount a high macrophage inflammatory reaction to a stimulus, and their BM cells strongly proliferate in response to the presence of IL-3. Thus, we examined whether or not the type of BM cell response to IL-3 (i.e., A/J- or C57BL/6-like) determines the magnitude of the macrophage inflammatory response using the A x B/B x A recombinant inbred (RI) mouse strain system. The two traits were found not to cosegregate, suggesting that they are not linked. The continuous strain distribution pattern of the magnitude of the macrophage inflammatory response obtained in mice of the A x B/B x A RI strains implies that this trait is under the control of several genes.