Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K+) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na+) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca2+) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na+ conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K+ channel selectivity in constitutive cell proliferation and hormone production.

The macrophage migration inhibitory factor (MIF) receptor (CD74) was cloned recently, but the signaling mechanism is not evident. We hypothesized that signaling requires an additional molecule such as CD44, which activates nonreceptor tyrosine kinases. We utilized the CD74- and CD44-deficient COS-7/M6 cell to create stable transfectants expressing CD74, CD44, and a truncated CD44 lacking its intracytoplasmic signaling domain. CD74 alone mediated MIF binding; however, MIF-induced ERK1 and ERK2 kinase phosphorylation required the coexpression of full-length CD44. MIF binding was associated with the serine phosphorylation of CD74 and CD44. Investigations that used siRNA or kinase inhibitors indicate that MIF-induced ERK1 and ERK2 activation through CD44 required the Src tyrosine kinase. Studies of CD74, CD44, and CD74-CD44 transformants and corresponding mutant cells showed that CD74 and CD44 were necessary for MIF protection from apoptosis. These data establish CD44 as an integral member of the CD74 receptor complex leading to MIF signal transduction.

Chemokines are the largest family of cytokines in human immunophysiology. These proteins are defined by four invariant cysteines and are categorized based on the sequence around the first two cysteines, which leads to two major and two minor subfamilies. Chemokines function by activating specific G protein-coupled receptors, which results in, among other functions, the migration of inflammatory and noninflammatory cells to the appropriate tissues or compartments within tissues. Some of these proteins and receptors have been implicated or shown to be involved in inflammation, autoimmune diseases, and infection by HIV-1. The three-dimensional structure of each monomer is virtually identical, but the quaternary structure of chemokines is different for each subfamily. Structure-function studies reveal several regions of chemokines to be involved in function, with the N-terminal region playing a dominant role. A number of proteins and small-molecule antagonists have been identified that inhibit chemokine activities. In this review, we discuss aspects of the structure, function, and inhibition of chemokines.

Macrophage migration inhibitory factor (MIF) is a pivotal regulator of the immune response. Neutralization or genetic deletion of MIF does not completely abrogate activation responses, however, and deletion of the MIF receptor, CD74, produces a more pronounced phenotype than MIF deficiency. We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a probable candidate to be the protein encoded by the homologous, D-dopachrome tautomerase (D-DT) gene. We show that recombinant D-DT protein binds CD74 with high affinity, leading to activation of ERK1/2 MAP kinase and downstream proinflammatory pathways. Circulating D-DT levels correlate with disease severity in sepsis or malignancy, and the specific immunoneutralization of D-DT protects mice from lethal endotoxemia by reducing the expression of downstream effector cytokines. These data indicate that D-DT is a MIF-like cytokine with an overlapping spectrum of activities that are important for our understanding of MIF-dependent physiology and pathology.inflammation | lipopolysaccharide | septic shock M acrophage migration inhibitory factor (MIF) is the first cytokine activity described and a key regulatory mediator that is released upon activation of different cell types (1-3). MIF increases macrophage antimicrobial responses and it is expressed upstream of cytokines such as tumor necrosis factor (TNF)-α, IFN-γ, and IL-1β (4). MIF activates immune cells by binding to CD74, leading to the recruitment of CD44 into a signaling complex, the stimulation of nonreceptor tyrosine kinases, and initiation of the ERK1/2 MAP kinase pathway (5, 6). The chemokine receptors CXCR2 and CXCR4 also become activated by MIF via noncognate interactions that are reinforced in the presence of CD74 (7). Among mesenchymal cell types, MIF binding to cardiomyocyte CD74 stimulates the AMP-activated kinase (AMPK) cascade to mediate protection from ischemic injury (8, 9).Although MIF receptor knockout mice (CD74) phenocopy features of MIF deficiency (10-12), recent observations have led to the hypothesis that there may be a second ligand for CD74. MIF-deficient B cells, for example, are more sensitive to apoptosis than wild-type B cells, but the magnitude of this defect is twofold more pronounced in CD74-deficient cells (13). Intravital microscopy studies also have shown a more pronounced effect of antagonism of CD74 than MIF in monocyte arrest (7). Finally, anti-MIF antibodies, although highly effective in experimental studies, do not completely inhibit CD74-dependent cellular activation responses (14).We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a likely candidate to be the protein encoded by the DDT gene, D-dopachrome tautomerase (D-DT). DDT and MIF show a conserved intron-exon structure and their coding regions are highly homologous. The genes for MIF and D-DT are in close apposition to each other and to two theta-class glutathione S-transferases, suggesting that these gene clusters arose by...

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6-A resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel a-helices that pack against a four-stranded a-sheet. The monomer has an additional two ,8-strands that interact with the a-sheets of adjacent subunits to form the interface between monomers. The three ,B-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.