Clues for New Therapeutics in Osteoporosis
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《新英格兰医药杂志》
More than 25 million people, most of whom are menopausal or elderly women, have a progressive loss of bone mass, which increases their susceptibility to fracture. The increasing prevalence and tremendous costs of this disorder underscore the importance of developing new treatments. Because the pathogenesis of osteoporosis is multifactorial — bone mass is influenced by physical activity, nutrition, and genetic factors — devising new therapeutics is a challenge. The recent findings of Klein et al. therefore come as welcome news: the authors identified an enzyme, 12/15-lipoxygenase, that contributes to natural variations in bone mass and skeletal development in mice, and showed that compounds that target this enzyme increase bone mass.1
Klein et al. used inbred mouse strains and genetic analyses to identify a region of chromosome 11 that influences bone mineral density. Substituting an 82-megabase stretch of this chromosome in the D2 strain of mice with the corresponding region from the B6 strain increased bone mineral density and femoral strength in the D2 mice. The investigators then narrowed the search to a stretch of about 31 megabases and found that, among the genes in this portion of the genome, only Alox15, which encodes 12/15-lipoxygenase, was differentially expressed in the two mouse strains. They then used engineered mice that completely lacked Alox15 and found that these mice had greater bone mineral density and stronger bones than wild-type mice. Mice carrying a single Alox15 allele had bones of intermediate strength, as compared with the knockout and wild-type mice. These findings demonstrate that the expression of 12/15-lipoxygenase is a key osteogenic regulator. Consistent with this observation are the findings that transient expression of 12/15-lipoxygenase in a stromal cell line reduces the biosynthesis of osteocalcin and alkaline phosphate and that, by oxygenating fatty acids, 12/15-lipoxygenase produces ligands, such as 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid (precursors of lipoxins), for peroxisome-proliferator–activated receptor (Figure 1). Activation of peroxisome-proliferator–activated receptor inhibits the differentiation of bone marrow precursors into osteoblasts.
Figure 1. Potential Mechanisms of the Effect of Lipoxygenase on Bone Mass.
The level of expression of 12/15-lipoxygenase in mice may induce variations in bone mass by initiating the synthesis of specific ligands that regulate osteogenesis, as indicated in the recent study by Klein et al.1 The enzyme 12/15-lipoxygenase is encoded on mouse chromosome 11, but in humans, this enzyme has a composite counterpart in the form of three separate enzymes: 12-lipoxygenase and 15-lipoxygenase type 1 and type 2. These nonheme iron-containing enzymes oxygenate the cis,cis-1,4-pentadiene units of polyunsaturated fatty acids by abstracting hydrogen and inserting molecular oxygen to generate hydroperoxide-containing intermediates that can be further transformed to bioactive ligands. These putative ligands may act directly on pathways dependent on peroxisome-proliferator–activated receptors, given the promiscuity of these receptors, or on surface receptors that can counterregulate the expression of specific cytokines and growth factors.2,3,4 Efforts to develop lipoxygenase-specific inhibitors that prevent diminutions in bone mass should focus on inhibitors that are specific for 15-lipoxygenase type 2, since this isoenzyme seems to aggravate bone loss in humans.
How might these findings help us to understand the pathogenesis of osteoporosis and develop treatments and strategies for prevention? It turns out that the mouse 12/15-lipoxygenase enzyme corresponds to at least three lipoxygenases in humans: 12-lipoxygenase is predominantly expressed in platelets and macrophages and is distinct from 15-lipoxygenase, which has two isoenzymes: type 1 (encoded by a gene at chromosome 17p13.3) and type 2 (encoded by a separate gene at 17p13.1).5
Although 15-lipoxygenase and 12-lipoxygenase are widely distributed in the plant and animal kingdoms, we know little of their roles in complex systems. We do, however, know that in the immune system, cell–cell interactions during host defense and inflammation provide the microenvironment for the synthesis of lipoxin through interactions between 15-lipoxygenase and 5-lipoxygenase and between 5-lipoxygenase and 12-lipoxygenase. The work of Klein et al. suggests that the known antiinflammatory properties of lipoxins, products of 15-lipoxygenase, reduce the expression of certain cytokines (interleukin-1 and tumor necrosis factor ) and growth factors that maintain osteoclast activity and that these lipoxins may therefore provide protection against cytokine-mediated bone loss. (In bone remodeling during menopausal osteoporosis, osteoblasts secrete higher-than-normal levels of cytokines, such as interleukin-1, tumor necrosis factor , and interleukin-6, as well as prostaglandins.) In addition, transgenic rabbits that overexpress human 15-lipoxygenase type 1 are protected against bone loss associated with periodontal disease.2 Thus, it seems possible that 15-lipoxygenase type 1 and type 2 may have antagonistic effects, with type 1 providing protection against bone loss through lipoxin-mediated suppression of specific cytokines, and type 2 aggravating bone loss.
The authors went on to test two inhibitors of 12/15-lipoxygenase in mouse models of osteoporosis and, in so doing, showed that interleukin-4 effects bone loss by increasing the expression of Alox15. Mice in which interleukin-4 is overexpressed typically have lower peak bone mass and bone strength than wild-type mice. After weaning, transgenic mice with overexpression of interleukin-4 were given feed containing one of the inhibitors. Twelve weeks later, these mice had greater bone mineral density and stronger bones than littermates that were given feed without an inhibitor. The other model of osteoporosis appropriately involved ovariectomy, which induces premature menopause. Ovariectomized mice that received feed containing an inhibitor lost less bone mass than did controls. At least one of these inhibitors is not believed to be particularly selective, meaning that it cannot differentiate between 15-lipoxygenase type 1 and type 2. Moreover, one of the inhibitors may simply act as an antioxidant.
These findings underscore the need to determine the types of cells, substrates, and metabolic pathways of lipid mediators3 that are altered in bone during osteoporosis. A better understanding of the function and expression of 12-lipoxygenase and 15-lipoxygenase isoenzymes type 1 and type 2 may hasten the development of novel antiosteoporotic drugs.
Source Information
From Brigham and Women's Hospital and Harvard University, Boston.
References
Klein RF, Allard J, Avnur Z, et al. Regulation of bone mass in mice by the lipoxygenase gene Alox15. Science 2004;303:229-232.
Serhan CN, Jain A, Marleau S, et al. Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators. J Immunol 2003;171:6856-6865.
Goh J, Godson C, Brady HR, Macmathuna P. Lipoxins: pro-resolution lipid mediators in intestinal inflammation. Gastroenterology 2003;124:1043-1054.
Kobayashi T, Narumiya S. Function of prostanoid receptors: studies on knockout mice. Prostaglandins Other Lipid Mediat 2002;68:557-573.
Krieg P, Marks F, Fürstenberger G. A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression. Genomics 2001;73:323-330.(Charles N. Serhan, Ph.D.)
Klein et al. used inbred mouse strains and genetic analyses to identify a region of chromosome 11 that influences bone mineral density. Substituting an 82-megabase stretch of this chromosome in the D2 strain of mice with the corresponding region from the B6 strain increased bone mineral density and femoral strength in the D2 mice. The investigators then narrowed the search to a stretch of about 31 megabases and found that, among the genes in this portion of the genome, only Alox15, which encodes 12/15-lipoxygenase, was differentially expressed in the two mouse strains. They then used engineered mice that completely lacked Alox15 and found that these mice had greater bone mineral density and stronger bones than wild-type mice. Mice carrying a single Alox15 allele had bones of intermediate strength, as compared with the knockout and wild-type mice. These findings demonstrate that the expression of 12/15-lipoxygenase is a key osteogenic regulator. Consistent with this observation are the findings that transient expression of 12/15-lipoxygenase in a stromal cell line reduces the biosynthesis of osteocalcin and alkaline phosphate and that, by oxygenating fatty acids, 12/15-lipoxygenase produces ligands, such as 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid (precursors of lipoxins), for peroxisome-proliferator–activated receptor (Figure 1). Activation of peroxisome-proliferator–activated receptor inhibits the differentiation of bone marrow precursors into osteoblasts.
Figure 1. Potential Mechanisms of the Effect of Lipoxygenase on Bone Mass.
The level of expression of 12/15-lipoxygenase in mice may induce variations in bone mass by initiating the synthesis of specific ligands that regulate osteogenesis, as indicated in the recent study by Klein et al.1 The enzyme 12/15-lipoxygenase is encoded on mouse chromosome 11, but in humans, this enzyme has a composite counterpart in the form of three separate enzymes: 12-lipoxygenase and 15-lipoxygenase type 1 and type 2. These nonheme iron-containing enzymes oxygenate the cis,cis-1,4-pentadiene units of polyunsaturated fatty acids by abstracting hydrogen and inserting molecular oxygen to generate hydroperoxide-containing intermediates that can be further transformed to bioactive ligands. These putative ligands may act directly on pathways dependent on peroxisome-proliferator–activated receptors, given the promiscuity of these receptors, or on surface receptors that can counterregulate the expression of specific cytokines and growth factors.2,3,4 Efforts to develop lipoxygenase-specific inhibitors that prevent diminutions in bone mass should focus on inhibitors that are specific for 15-lipoxygenase type 2, since this isoenzyme seems to aggravate bone loss in humans.
How might these findings help us to understand the pathogenesis of osteoporosis and develop treatments and strategies for prevention? It turns out that the mouse 12/15-lipoxygenase enzyme corresponds to at least three lipoxygenases in humans: 12-lipoxygenase is predominantly expressed in platelets and macrophages and is distinct from 15-lipoxygenase, which has two isoenzymes: type 1 (encoded by a gene at chromosome 17p13.3) and type 2 (encoded by a separate gene at 17p13.1).5
Although 15-lipoxygenase and 12-lipoxygenase are widely distributed in the plant and animal kingdoms, we know little of their roles in complex systems. We do, however, know that in the immune system, cell–cell interactions during host defense and inflammation provide the microenvironment for the synthesis of lipoxin through interactions between 15-lipoxygenase and 5-lipoxygenase and between 5-lipoxygenase and 12-lipoxygenase. The work of Klein et al. suggests that the known antiinflammatory properties of lipoxins, products of 15-lipoxygenase, reduce the expression of certain cytokines (interleukin-1 and tumor necrosis factor ) and growth factors that maintain osteoclast activity and that these lipoxins may therefore provide protection against cytokine-mediated bone loss. (In bone remodeling during menopausal osteoporosis, osteoblasts secrete higher-than-normal levels of cytokines, such as interleukin-1, tumor necrosis factor , and interleukin-6, as well as prostaglandins.) In addition, transgenic rabbits that overexpress human 15-lipoxygenase type 1 are protected against bone loss associated with periodontal disease.2 Thus, it seems possible that 15-lipoxygenase type 1 and type 2 may have antagonistic effects, with type 1 providing protection against bone loss through lipoxin-mediated suppression of specific cytokines, and type 2 aggravating bone loss.
The authors went on to test two inhibitors of 12/15-lipoxygenase in mouse models of osteoporosis and, in so doing, showed that interleukin-4 effects bone loss by increasing the expression of Alox15. Mice in which interleukin-4 is overexpressed typically have lower peak bone mass and bone strength than wild-type mice. After weaning, transgenic mice with overexpression of interleukin-4 were given feed containing one of the inhibitors. Twelve weeks later, these mice had greater bone mineral density and stronger bones than littermates that were given feed without an inhibitor. The other model of osteoporosis appropriately involved ovariectomy, which induces premature menopause. Ovariectomized mice that received feed containing an inhibitor lost less bone mass than did controls. At least one of these inhibitors is not believed to be particularly selective, meaning that it cannot differentiate between 15-lipoxygenase type 1 and type 2. Moreover, one of the inhibitors may simply act as an antioxidant.
These findings underscore the need to determine the types of cells, substrates, and metabolic pathways of lipid mediators3 that are altered in bone during osteoporosis. A better understanding of the function and expression of 12-lipoxygenase and 15-lipoxygenase isoenzymes type 1 and type 2 may hasten the development of novel antiosteoporotic drugs.
Source Information
From Brigham and Women's Hospital and Harvard University, Boston.
References
Klein RF, Allard J, Avnur Z, et al. Regulation of bone mass in mice by the lipoxygenase gene Alox15. Science 2004;303:229-232.
Serhan CN, Jain A, Marleau S, et al. Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators. J Immunol 2003;171:6856-6865.
Goh J, Godson C, Brady HR, Macmathuna P. Lipoxins: pro-resolution lipid mediators in intestinal inflammation. Gastroenterology 2003;124:1043-1054.
Kobayashi T, Narumiya S. Function of prostanoid receptors: studies on knockout mice. Prostaglandins Other Lipid Mediat 2002;68:557-573.
Krieg P, Marks F, Fürstenberger G. A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression. Genomics 2001;73:323-330.(Charles N. Serhan, Ph.D.)