TiPS - February 1992 IVoI. 131
45
Lipocortin-1exhibitsnovel actions, providingclinicalopportunities Lipocortin-1 (annexin-1) has been of interest to scientists from a wide range of disciplines, but an understanding of the functions, control cf synthesis and mechanisms of action of this protein has been confounded by its diversity of actions and by a number of technical and experimental problems. Recent findings, summarized at a meeting on the biology of lipocortin-1* have significantly advanced our understanding of this complex molecule and revealed novel actions that may be of direct clinical relevance. Lipocortin-1, a member of the ‘annexin’ family of Ca*+- and phospholipid-binding proteins, was originally identified as an endogenous mediator of the antiinflammatory actions of glucocorticoids. Data obtained independently by several research groups using the highly purified recombinant protein have confirmed these actions. For example, the recombinant human molecule potently inhibits eicosanoid production, replication of A549 cells, carrageenin oedema and cytokinemigration induced neutrophil (Rod Flower, William Harvey Institute, London), Research chemotaxis (Franqoise RussoMarie, INSERM, Paris) and fever (Nancy Rothwell, University of Manchester). However, perhaps more significant are observations that neutralizing anti-lipocortin-1 antibodies reverse anti-inflammatory (Flower) and antipyretic (Rothwell) actions of glucocorticoids in vivo, indicating that endogenous lipocortin-1 mediates these effects. Induction of lipocortin-1 expression by glucocorticoids has been reported in various cell types (Flower) and in macrophages from patients undergoing steroid treatment (Luca Parente, Sclavo Research Institute, Siena; Nick Bath Institute of Goulding, Rheumatic Diseases). However, *Biology of tipocortin-2, Royal Society London, 25 November 2991.
lipocortin-1 expression appears to be independent of glucocorticoid status in certain cell lines (Jeff Browning, Biogen Research Corporation, Boston) and in the brain (Julia Buckingham, Charing Cross Hospital Medical School, London). Studies by Parente indicate that glucocorticoid responsiveness may be dependent on the stage of cell differentiation. It is apparent that not all actions of glucocorticoids are mediated by lipocortin-1 (Flower), and that its expression is additionally controlled by a number of other factors. Harry Haigler (University of California, Irvine) reported that the hormone prolactin, which causes differentiation of the pigeon crop sac, also induces lipocortin-1. Haigler identified an additional lipocortin-l-like molecule (P35) in this organ that
was prolactin-independent: interestingly this molecule was not a substrate for growth factor kinases. Lipocortin-1 exists primarily as an intracellular protein, and lacks a leader sequence necessary for cellular release by classical mechanisms. Studies by Browning on cell lines stably transfected with the human lipocortin-1 gene, have shown that the protein exists on the outer surface of the cell in two forms, one of which may be detached by EDTA and another, of greater abundance, tbtt is EDTA resistant. Surface forms of lipocortin were observed on several cell lines, indicating that this may be a physiologically important pool. Since lipocortin-1 appears to act in a predominantly autocrine manner, it is possible that detection of the protein in extracellular flmds results from release by damaged cells. However, Haigler presented evidence that lipocortin-1 can be secreted from cells, since it is found in exceptionally high concentrations in human seminal fluid, apparently originating from the prostate gland. The lipocortin-1 content
Fig. 1. Photomicrogaph of lipocortin-1 immuno~actfvity in frontal sections of the rat hypothalamus. A: posterior part of the median eminence. showing a h&h density of immunoreactivenerve fibrea (F) and tanycy7esWeciaC ized ependymal cells). B: middle region of the median eminence, with a similarty dense nehvorir of immunomactive fib= From Strijbos, P. J. L. M. et a/. (1991) Brain Res. 553,24%260.
Q 1992, Eltevier Science Publishers Ltd (UK)
TiPS - February 1992 [Vol. 231
46 of rat prost??e
is increased by severalfoli following castration, and this effect is reversed by testosterone. The function of lipomrtin-1 in seminal fluid is unknown, but the presence of such an anti-inflammatory and immunosuppressive molecule might play a role in facilitating fertilization or implantation. exerts several Lipocortin-1 actions in the brain, where it is expressed in neurons and glia (see Fii. 1). A biologically active recombinant fragment of lipocortin-1 inhibits the central actions of cytokines on fever and thermogenesis, and has potent neuroprotective actions against ischaemic and excitotoxic damage in the rat (Rothwell). Lipocortin-1 expression in brain is increased in response to many forms of damage, and administration to rats of anti-lipocortin-1 antibody exacerbates brain damage, indicating that lipocortin-1 acts as an endogenous neuroprotective agent. Complex neumen doaine actions of lipocortin-1 on the hypothalamic-pituitary axis were reported
by Buckingham. It inhibits hypothalamic release of corticotrophinreleasing factor but stimulates release of arginine vasopressin. In the rat pituitary, dexamethasone and lipocortin-1 inhibit the release of ACTH and prolactin, and the anti-lipocortin-1 neutralizing antibody reverses the dexamethasone effect. This suggests a role for the protein in control of the release of some pituitary hormones. The first evidence of a cellular ‘receptor’ for lipocortin-1 was reported by Goulding, who has identified a Ca2’-dependent binding site on human peripheral blood leukocytes, ranging in density from 1000 (lymphocytes) to 100000 (monocytes) per cell. A marked decrease in the density of binding sites was observed in patients with rheumatoid arthritis. Preliminary data presented indicate the presence of two specific 15 and 18 kDa binding proteins, and further characterization may reveal the structure of a putative receptor for lipocortin-1. As yet little is known of the mechanisms of these diverse
Nucleoside diphosphate kinases as potential new targets for control of development and cancer Recently, converging data from several laboratories have led to the identification of the products of the genes nm23, involved in mammalian tumor malignancy’, and awd, crucial for Drosophila developmentz*3, as nucleoside diphosphate (NDI?) kinases4. These findings led to renewed interest in NDP kinases, longconsidered ‘housekeeping’ enzymes, and their possible roles in cellular regulation and the control of the metastatic potential of tumors. This article summarizes these results in the light of the proposed role of NDP kinases in GTP-controlled processes5*6(Fig. 1). NDP kinases (EC 2.7.4.6) are ubiquitous and essential for the synthesis of nucleoside triphos@ 1992. Elaevier Sdence Publishem Ltd (UK)
phates other than ATP (Ref. 7). They catalyse phosphorylation of nucleoside 5’-diphosphates to corresponding triphosphates by a ping-pong mechanism with formation of high-energy phosphohistidine intermediates. They are found mainly in the cytosol, but also in the intermembrane space of mitochondria and, in minor quantities, associated with plasma membranes and nuclei. Eukaryotic NDP kinases are hexamers, forming a family of isoenzymes that exhibits a marked isoelectric variability. Recent studies have shown that the NDP kinase purified from human erythrocytes consists of two types of monomers, A and B, which are highly homologous (88% identity) but
actions of lipocortin-1. The protein is known to bind Ca2+ and anionic phospholipids and, through this or other mechanisms, to modulate phospholipase A2 activity (thus inhibiting arachidonic acid release and subsequent eicosanoid synthesis). It can act as a substrate for the EGF receptor kinase, influence vesicle fusion and therefore transmitter release and, in some cell systems, may directly form ion channels. It seems unlikely that the varied effects of lipocortin-1 will be related to a single mechanism of action. However, the production of biologically active recombinant forms of lipocortin-1 and neutralizing antibodies, and identification of defined systems that exhibit biological responses to the protein, should now allow rapid progress towards understanding the biology of this tantalizing molecule. NANCY J. ROTHWELL
AND ROD FLOWRR*
Department of Physiological Sciences, University of Manchester, Manchester Ml3 9PT, UK and Williarr~ Harvey Research Institute, St Bartholomew’s Hospital Medical College, LondonEClM 684, UK.
have different p1 values. The random association of the A and B polypeptides could account for the isozyme patterns found in various cell types’s. cDNAs encoding NDP kinases from various organisms have recently been sequenced (listed in Ref. 8). The mammalian nm23 and Drosophila awd genes13 were isolated on the basis of their association with a particular phenotype and were shown a posteriori to encode NDP kinases (see below). All of these proteins are highly conserved (44% identity between bacterial and human enzymes), Pointing to a strong selection pressure for structural and functional conservation. NDP kinases in cancer and development The nm23 cDNA was originally identified by differential screening of a cDNA library with mRNA isolated from murine melanoma cell lines of low and high metastatic potential’. Reduced expression of the nm23 gene was correlated with a high metastatic potential in certain rodent model
45
Lipocortin-1exhibitsnovel actions, providingclinicalopportunities Lipocortin-1 (annexin-1) has been of interest to scientists from a wide range of disciplines, but an understanding of the functions, control cf synthesis and mechanisms of action of this protein has been confounded by its diversity of actions and by a number of technical and experimental problems. Recent findings, summarized at a meeting on the biology of lipocortin-1* have significantly advanced our understanding of this complex molecule and revealed novel actions that may be of direct clinical relevance. Lipocortin-1, a member of the ‘annexin’ family of Ca*+- and phospholipid-binding proteins, was originally identified as an endogenous mediator of the antiinflammatory actions of glucocorticoids. Data obtained independently by several research groups using the highly purified recombinant protein have confirmed these actions. For example, the recombinant human molecule potently inhibits eicosanoid production, replication of A549 cells, carrageenin oedema and cytokinemigration induced neutrophil (Rod Flower, William Harvey Institute, London), Research chemotaxis (Franqoise RussoMarie, INSERM, Paris) and fever (Nancy Rothwell, University of Manchester). However, perhaps more significant are observations that neutralizing anti-lipocortin-1 antibodies reverse anti-inflammatory (Flower) and antipyretic (Rothwell) actions of glucocorticoids in vivo, indicating that endogenous lipocortin-1 mediates these effects. Induction of lipocortin-1 expression by glucocorticoids has been reported in various cell types (Flower) and in macrophages from patients undergoing steroid treatment (Luca Parente, Sclavo Research Institute, Siena; Nick Bath Institute of Goulding, Rheumatic Diseases). However, *Biology of tipocortin-2, Royal Society London, 25 November 2991.
lipocortin-1 expression appears to be independent of glucocorticoid status in certain cell lines (Jeff Browning, Biogen Research Corporation, Boston) and in the brain (Julia Buckingham, Charing Cross Hospital Medical School, London). Studies by Parente indicate that glucocorticoid responsiveness may be dependent on the stage of cell differentiation. It is apparent that not all actions of glucocorticoids are mediated by lipocortin-1 (Flower), and that its expression is additionally controlled by a number of other factors. Harry Haigler (University of California, Irvine) reported that the hormone prolactin, which causes differentiation of the pigeon crop sac, also induces lipocortin-1. Haigler identified an additional lipocortin-l-like molecule (P35) in this organ that
was prolactin-independent: interestingly this molecule was not a substrate for growth factor kinases. Lipocortin-1 exists primarily as an intracellular protein, and lacks a leader sequence necessary for cellular release by classical mechanisms. Studies by Browning on cell lines stably transfected with the human lipocortin-1 gene, have shown that the protein exists on the outer surface of the cell in two forms, one of which may be detached by EDTA and another, of greater abundance, tbtt is EDTA resistant. Surface forms of lipocortin were observed on several cell lines, indicating that this may be a physiologically important pool. Since lipocortin-1 appears to act in a predominantly autocrine manner, it is possible that detection of the protein in extracellular flmds results from release by damaged cells. However, Haigler presented evidence that lipocortin-1 can be secreted from cells, since it is found in exceptionally high concentrations in human seminal fluid, apparently originating from the prostate gland. The lipocortin-1 content
Fig. 1. Photomicrogaph of lipocortin-1 immuno~actfvity in frontal sections of the rat hypothalamus. A: posterior part of the median eminence. showing a h&h density of immunoreactivenerve fibrea (F) and tanycy7esWeciaC ized ependymal cells). B: middle region of the median eminence, with a similarty dense nehvorir of immunomactive fib= From Strijbos, P. J. L. M. et a/. (1991) Brain Res. 553,24%260.
Q 1992, Eltevier Science Publishers Ltd (UK)
TiPS - February 1992 [Vol. 231
46 of rat prost??e
is increased by severalfoli following castration, and this effect is reversed by testosterone. The function of lipomrtin-1 in seminal fluid is unknown, but the presence of such an anti-inflammatory and immunosuppressive molecule might play a role in facilitating fertilization or implantation. exerts several Lipocortin-1 actions in the brain, where it is expressed in neurons and glia (see Fii. 1). A biologically active recombinant fragment of lipocortin-1 inhibits the central actions of cytokines on fever and thermogenesis, and has potent neuroprotective actions against ischaemic and excitotoxic damage in the rat (Rothwell). Lipocortin-1 expression in brain is increased in response to many forms of damage, and administration to rats of anti-lipocortin-1 antibody exacerbates brain damage, indicating that lipocortin-1 acts as an endogenous neuroprotective agent. Complex neumen doaine actions of lipocortin-1 on the hypothalamic-pituitary axis were reported
by Buckingham. It inhibits hypothalamic release of corticotrophinreleasing factor but stimulates release of arginine vasopressin. In the rat pituitary, dexamethasone and lipocortin-1 inhibit the release of ACTH and prolactin, and the anti-lipocortin-1 neutralizing antibody reverses the dexamethasone effect. This suggests a role for the protein in control of the release of some pituitary hormones. The first evidence of a cellular ‘receptor’ for lipocortin-1 was reported by Goulding, who has identified a Ca2’-dependent binding site on human peripheral blood leukocytes, ranging in density from 1000 (lymphocytes) to 100000 (monocytes) per cell. A marked decrease in the density of binding sites was observed in patients with rheumatoid arthritis. Preliminary data presented indicate the presence of two specific 15 and 18 kDa binding proteins, and further characterization may reveal the structure of a putative receptor for lipocortin-1. As yet little is known of the mechanisms of these diverse
Nucleoside diphosphate kinases as potential new targets for control of development and cancer Recently, converging data from several laboratories have led to the identification of the products of the genes nm23, involved in mammalian tumor malignancy’, and awd, crucial for Drosophila developmentz*3, as nucleoside diphosphate (NDI?) kinases4. These findings led to renewed interest in NDP kinases, longconsidered ‘housekeeping’ enzymes, and their possible roles in cellular regulation and the control of the metastatic potential of tumors. This article summarizes these results in the light of the proposed role of NDP kinases in GTP-controlled processes5*6(Fig. 1). NDP kinases (EC 2.7.4.6) are ubiquitous and essential for the synthesis of nucleoside triphos@ 1992. Elaevier Sdence Publishem Ltd (UK)
phates other than ATP (Ref. 7). They catalyse phosphorylation of nucleoside 5’-diphosphates to corresponding triphosphates by a ping-pong mechanism with formation of high-energy phosphohistidine intermediates. They are found mainly in the cytosol, but also in the intermembrane space of mitochondria and, in minor quantities, associated with plasma membranes and nuclei. Eukaryotic NDP kinases are hexamers, forming a family of isoenzymes that exhibits a marked isoelectric variability. Recent studies have shown that the NDP kinase purified from human erythrocytes consists of two types of monomers, A and B, which are highly homologous (88% identity) but
actions of lipocortin-1. The protein is known to bind Ca2+ and anionic phospholipids and, through this or other mechanisms, to modulate phospholipase A2 activity (thus inhibiting arachidonic acid release and subsequent eicosanoid synthesis). It can act as a substrate for the EGF receptor kinase, influence vesicle fusion and therefore transmitter release and, in some cell systems, may directly form ion channels. It seems unlikely that the varied effects of lipocortin-1 will be related to a single mechanism of action. However, the production of biologically active recombinant forms of lipocortin-1 and neutralizing antibodies, and identification of defined systems that exhibit biological responses to the protein, should now allow rapid progress towards understanding the biology of this tantalizing molecule. NANCY J. ROTHWELL
AND ROD FLOWRR*
Department of Physiological Sciences, University of Manchester, Manchester Ml3 9PT, UK and Williarr~ Harvey Research Institute, St Bartholomew’s Hospital Medical College, LondonEClM 684, UK.
have different p1 values. The random association of the A and B polypeptides could account for the isozyme patterns found in various cell types’s. cDNAs encoding NDP kinases from various organisms have recently been sequenced (listed in Ref. 8). The mammalian nm23 and Drosophila awd genes13 were isolated on the basis of their association with a particular phenotype and were shown a posteriori to encode NDP kinases (see below). All of these proteins are highly conserved (44% identity between bacterial and human enzymes), Pointing to a strong selection pressure for structural and functional conservation. NDP kinases in cancer and development The nm23 cDNA was originally identified by differential screening of a cDNA library with mRNA isolated from murine melanoma cell lines of low and high metastatic potential’. Reduced expression of the nm23 gene was correlated with a high metastatic potential in certain rodent model
