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Generation of Soluble Leptin Receptor by Ectodomain Shedding of Membrane-spanning Receptors in Vitro and in Vivo

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m205825200

ISSN

1083-351X

Autores

Hongfei Ge, Lu Huang, Tiffany Pourbahrami, Cai Li,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

Leptin is an adipocyte-derived hormone with potent effects on food intake and body weight. Genetically obese rodents with mutations of leptin or leptin receptor develop morbid obesity and diabetes. The receptor for leptin, OB-R, is alternatively spliced to at least five transcripts, encoding receptors designated OB-Ra, -b, -c, -d, and -e. OB-Re does not encode a transmembrane domain and is secreted. In humans, transcripts corresponding to OB-Re have not been discovered. However, soluble leptin receptor does circulate in human plasma and represents the major leptin-binding activity. In this report, we attempted to determine whether the soluble leptin receptor may also be derived from membrane-spanning receptor isoforms by ectodomain shedding. Using stable cell lines expressing both OB-Ra, the most abundant leptin receptor isoform, and OB-Rb, the signaling form of the leptin receptor, we demonstrate that soluble leptin receptor protein can indeed be generated by proteolytic cleavage of these two receptor isoforms in vitro. Experiments using adenoviruses expressing dually tagged OB-Ra or Ob-Rb also demonstrate that soluble leptin receptor may be derived from ectodomain shedding of both receptor isoforms in vivo. Because our earlier and other studies have shown that the soluble receptors modulate the levels as well as activity of leptin, our findings suggest that regulated shedding of the ectodomain of membrane-spanning leptin receptors may represent a novel mechanism of modulating leptin's biological activity. Leptin is an adipocyte-derived hormone with potent effects on food intake and body weight. Genetically obese rodents with mutations of leptin or leptin receptor develop morbid obesity and diabetes. The receptor for leptin, OB-R, is alternatively spliced to at least five transcripts, encoding receptors designated OB-Ra, -b, -c, -d, and -e. OB-Re does not encode a transmembrane domain and is secreted. In humans, transcripts corresponding to OB-Re have not been discovered. However, soluble leptin receptor does circulate in human plasma and represents the major leptin-binding activity. In this report, we attempted to determine whether the soluble leptin receptor may also be derived from membrane-spanning receptor isoforms by ectodomain shedding. Using stable cell lines expressing both OB-Ra, the most abundant leptin receptor isoform, and OB-Rb, the signaling form of the leptin receptor, we demonstrate that soluble leptin receptor protein can indeed be generated by proteolytic cleavage of these two receptor isoforms in vitro. Experiments using adenoviruses expressing dually tagged OB-Ra or Ob-Rb also demonstrate that soluble leptin receptor may be derived from ectodomain shedding of both receptor isoforms in vivo. Because our earlier and other studies have shown that the soluble receptors modulate the levels as well as activity of leptin, our findings suggest that regulated shedding of the ectodomain of membrane-spanning leptin receptors may represent a novel mechanism of modulating leptin's biological activity. Leptin is an adipocyte-derived hormone of 167 amino acids (1Zhang Y. Proenca P. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11631) Google Scholar). It has potent weight-reducing effects in vivo (2Campfield L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3060) Google Scholar, 3Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4199) Google Scholar, 4Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3850) Google Scholar). Inob/ob mice, the gene encoding leptin is mutated, resulting in morbid obesity and associated abnormalities, including hyperphagia, hypothermia, diabetes, and infertility.The leptin receptor, OB-R, is a member of the cytokine receptor family (5Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3205) Google Scholar). It is encoded by the diabetes (db) gene, mutation of which also results in phenotypes similar to that exhibited byob/ob mice. OB-R is alternatively spliced into at least five transcripts from a single gene. These transcripts encode proteins that are called the long (OB-Rb), short (OB-Ra, -c, and -d), and soluble (OB-Re) forms of the leptin receptor. With the exception of the soluble leptin receptor, other receptor isoforms differ from each other by the alternative use of a unique terminal coding exon (6Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2095) Google Scholar). OB-Rb is essential in mediating leptin's weight-reducing effects via the hypothalamus (6Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2095) Google Scholar, 7Chen H. Charlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R.I. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1919) Google Scholar).OB-R is expressed in both the nervous system and in peripheral tissues. The relative expression levels of different receptor isoforms vary among tissues, possibly to allow leptin's biological activity to be more precisely regulated at various leptin target sites (8Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (730) Google Scholar). OB-Rb is enriched in the hypothalamus, the site of leptin's action on food intake and body weight. Leptin activation of OB-Rb within this brain region results in the inhibition of neuropeptide Y/agouti-related protein neurons and activation of pro-opiomelanocortin neurons (9Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (626) Google Scholar). Leptin-activated pro-opiomelanocortin neurons become depolarized and release anorexigenic peptides; leptin-inhibited neuropeptide Y/agouti-related protein neurons become hyperpolarized and reduce the release of orexigenic peptides (10Cowley M.A. Smart J.L. Rubinstein M. Cerdan M.G. Diano S. Horvath T.L. Cone R.D. Low M.J. Nature. 2001; 411: 480-484Crossref PubMed Scopus (1754) Google Scholar). These neural circuits represent the main known downstream mediators of leptin's biological effect on food intake. OB-Rb can also activate signal transduction in a variety of peripheral tissues, including adipose tissue, T cells, endothelial cells, and pancreatic β-cells (11Kim Y.B. Uotani S. Pierroz D.D. Flier J.S. Kahn B.B. Endocrinology. 2000; 141: 2328-2339Crossref PubMed Scopus (196) Google Scholar, 12Lord G.M. Matarese G. Howard J.K. Baker R.J. Bloom S.R. Lechler R.I. Nature. 1998; 394: 897-901Crossref PubMed Scopus (1828) Google Scholar, 13Sierra-Honigmann M.R. Nath A.K. Murakami C. Garcia-Cardena G. Papapetropoulos A. Sessa W.C. Madge L.A. Schechner J.S. Schwabb M.B. Polverini P.J. Flores-Riveros J.R. Science. 1998; 281: 1683-1686Crossref PubMed Scopus (1266) Google Scholar, 14Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (372) Google Scholar, 15Huang L. Li C. Cell Res. 2000; 10: 81-92Crossref PubMed Scopus (187) Google Scholar). More recently, it is also demonstrated that leptin-induced fatty acid oxidation in muscle via 5′-AMP-activated protein kinase is mediated by both leptin acting on muscle directly and by functioning through the hypothalamic-sympathetic nervous system axis (16Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1651) Google Scholar). Taken together, these results confirm that direct as well as indirect leptin signaling at these sites may be necessary for the many biological effects of leptin. In all cases, the presence of OB-Rb is required, asdb/db mice that are without this receptor isoform do not respond to leptin.Although OB-Rb is essential in mediating leptin's biological effects, other receptor isoforms may still be necessary for leptin to exert its full spectrum of in vivo functions. Among the short forms of the leptin receptor, OB-Ra is most abundantly expressed (17Fei H. Okano H.J. Li C. Lee G.-H. Zhao C. Darnell R. Friedman J.M. Prod. Natl. Acad. Sci., U. S. A. 1997; 94: 7001-7005Crossref PubMed Scopus (652) Google Scholar). It is enriched at the choroid plexus and brain microvessels, sites of the blood-cerebrospinal fluid barrier and the blood-brain barrier, suggesting that it may be involved in the transport of leptin across these barriers to reach the hypothalamus.Previously, we have shown that the secreted form of the leptin receptor, OB-Re, circulates in mouse plasma and is capable of binding to leptin (18Li C. Ioffe E. Fidahusein N. Connolly E. Friedman J.M. J. Biol. Chem. 1998; 273: 10078-10082Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In this report, we present evidence that circulating soluble leptin receptor may arise from both OB-Re mRNA and ectodomain shedding of membrane-spanning OB-R isoforms. We demonstrate that stable cell lines overexpressing OB-Ra or OB-Rb are capable of releasing soluble leptin receptor into the media by proteolytic cleavage, as detected by leptin-Sepharose pull-down assay. Furthermore, we demonstrate that adenovirus-mediated in vivooverexpression of OB-Ra or OB-Rb also results in release of soluble leptin receptor into plasma in mice via the same mechanism. Ectodomain shedding of both OB-Ra and OB-Rb is detected by a monoclonal antibody recognizing the FLAG epitope tag fused in-frame with the amino terminus of recombinant receptor protein. Stable 293 cells expressing OB-Ra and OB-Rb release the soluble leptin receptor under serum-free conditions, suggesting that the cleavage of membrane-bound receptor may be mediated by proteases resident in the cell. Because the soluble leptin receptor affects leptin's effect on food intake and body weight inob/ob mice (19Huang L. Wang Z. Li C. J. Biol. Chem. 2001; 276: 6343-6349Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), regulated ectodomain shedding of membrane-spanning leptin receptors may represent a novel mechanism of regulating leptin's biological activity.DISCUSSIONIn our earlier studies, we demonstrated that the soluble leptin receptor circulates in plasma and is capable of binding to leptin (18Li C. Ioffe E. Fidahusein N. Connolly E. Friedman J.M. J. Biol. Chem. 1998; 273: 10078-10082Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Our recently published results also demonstrated that the soluble receptor might play key roles in determining the amount of total leptin in circulation (19Huang L. Wang Z. Li C. J. Biol. Chem. 2001; 276: 6343-6349Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Other studies also suggest that expression and plasma concentration of the soluble leptin receptor may be regulated (29Gavrilova O. Barr V. Marcus-Samuels B. Reitman M. J. Biol. Chem. 1997; 272: 30546-30551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 30Widjaja A. Kielstein J.T. Horn R. von Zur Muhlen A. Kliem V. Brabant G. Nephrol. Dial. Transplant. 2000; 15: 846-850Crossref PubMed Scopus (41) Google Scholar). However, the origin of circulating soluble leptin receptor in plasma has not been clearly defined, especially as the mRNA splice variant encoding this receptor isoform in humans has not been found (26Chua Jr., S.C. Koutras I.K. Han L. Liu S.M. Kay J. Young S.J. Chung W.K. Leibel R.L. Genomics. 1997; 45: 264-270Crossref PubMed Scopus (125) Google Scholar).Because no post-translational modification of leptin occurs in vivo (31Cohen S.L. Halaas J.L. Friedman J.M. Chait B.T. Bennett L. Chang D. Hecht R. Collins F. Nature. 1996; 382: 589Crossref PubMed Scopus (91) Google Scholar), soluble leptin receptor may be an important factor regulating leptin's availability and bioactivity. This hypothesis is supported by findings that in human plasma, soluble leptin receptor represents the major leptin binding activity (27Lammert A. Kiess W. Bottner A. Glasow A. Kratzsch J. Biochem. Biophys. Res. Commun. 2001; 283: 982-988Crossref PubMed Scopus (243) Google Scholar).The main site of expression of the mRNA splice variant encoding the soluble leptin receptor in vivo remains unknown. Previously, we failed to detect a signal for OB-Re when Northern blot analysis was performed (17Fei H. Okano H.J. Li C. Lee G.-H. Zhao C. Darnell R. Friedman J.M. Prod. Natl. Acad. Sci., U. S. A. 1997; 94: 7001-7005Crossref PubMed Scopus (652) Google Scholar). Available data have demonstrated that in mice, OB-Re is expressed by the placenta. Its expression starts at day 14 of pregnancy, peaking just before parturition to about 40-fold the level found in non-pregnant mice (29Gavrilova O. Barr V. Marcus-Samuels B. Reitman M. J. Biol. Chem. 1997; 272: 30546-30551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 32Yamaguchi M. Murakami T. Yasui Y. Otani S. Kawai M. Kishi K. Kurachi H. Shima K. Aono T. Murata Y. Biochem. Biophys. Res. Commun. 1998; 252: 363-367Crossref PubMed Scopus (24) Google Scholar). In rats and humans, the pregnancy-associated rise of circulating leptin and its soluble receptor is relatively modest, achieving only a 2-fold increaseversus a more than 40-fold increase in mice (33Lewandowski K. Horn R. O'Callaghan C.J. Dunlop D. Medley G.F. O'Hare P. Brabant G. J. Clin. Endocrinol. Metab. 1999; 84: 300-306Crossref PubMed Scopus (130) Google Scholar).Our current study provided evidence of an additional mechanism for the generation of soluble leptin receptor by ectodomain shedding of membrane-spanning receptor, both in vitro and in vivo. This result is also in agreement with an earlier report on the generation of human soluble leptin receptor from transiently expressed membrane-spanning receptors in tissue culture (20Maamra M. Bidlingmaier M. Postel-Vinay M.C. Wu Z. Strasburger C.J. Ross R.J. Endocrinology. 2001; 142: 4389-4393Crossref PubMed Scopus (106) Google Scholar).Ectodomain shedding by proteolysis to yield soluble intercellular regulators has been observed for many proteins, such as tumor necrosis factor α and transforming growth factor α (34Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1356) Google Scholar). The responsible protease for these proteins, tumor necrosis factor α-converting enzyme, is a member of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinases. Molecules that are capable of inhibiting metalloproteinases are being tested as promising reagents for cancer therapy (35Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2349) Google Scholar). At the present time, the modulation of the soluble leptin receptor on leptin biology is not well understood. Initial evidence of the presence of free and bound leptin in human circulation was obtained by gel-filtration chromatography of plasma containing added 125I-leptin tracers (36Sinha M.K. Opentanova I. Ohannesian J.P. Kolaczynski J.W. Heiman M.L. Hale J. Becker G.W. Bowsher R.R. Stephens T.W. Caro J.F. J. Clin. Invest. 1996; 98: 1277-1282Crossref PubMed Scopus (515) Google Scholar). This and later studies showed that obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans, whereas weight loss increases it (37Laimer M. Ebenbichler C.F. Kaser S. Sandhofer A. Weiss H. Nehoda H. Aigner F. Patsch J.R. Obes. Res. 2002; 10: 597-601Crossref PubMed Scopus (74) Google Scholar, 38van Dielen F.M. van 't Veer C. Buurman W.A. Greve J.W. J. Clin. Endocrinol. Metab. 2002; 87: 1708-1716Crossref PubMed Scopus (117) Google Scholar, 39Lahlou N. Issad T. Lebouc Y. Carel J.C. Camoin L. Roger M. Girard J. Diabetes. 2002; 51: 1980-1985Crossref PubMed Scopus (35) Google Scholar, 40Ogier V. Ziegler O. Mejean L. Nicolas J.P. Stricker-Krongrad A. Int. J. Obes. Relat. Metab. Disord. 2002; 26: 496-503Crossref PubMed Scopus (71) Google Scholar). In others studies, obese and normal weight individuals were found to have similar amount of total circulating soluble leptin receptor, yet in the obese most soluble leptin receptor is bound to leptin, whereas in lean individuals a much smaller percentage of soluble leptin receptor is bound to leptin (75% in the obese versus 33% in the lean); thus, the inability to up-regulate circulating soluble leptin receptor could be a factor in the pathogenesis of obesity (36Sinha M.K. Opentanova I. Ohannesian J.P. Kolaczynski J.W. Heiman M.L. Hale J. Becker G.W. Bowsher R.R. Stephens T.W. Caro J.F. J. Clin. Invest. 1996; 98: 1277-1282Crossref PubMed Scopus (515) Google Scholar, 38van Dielen F.M. van 't Veer C. Buurman W.A. Greve J.W. J. Clin. Endocrinol. Metab. 2002; 87: 1708-1716Crossref PubMed Scopus (117) Google Scholar, 41Wu Z. Bidlingmaier M. Liu C. De Souza E.B. Tschop M. Morrison K.M. Strasburger C.J. J. Clin. Endocrinol. Metab. 2002; 87: 2931-2939Crossref PubMed Scopus (24) Google Scholar). However, a larger percentage of leptin circulates in the free form in the obese (41Wu Z. Bidlingmaier M. Liu C. De Souza E.B. Tschop M. Morrison K.M. Strasburger C.J. J. Clin. Endocrinol. Metab. 2002; 87: 2931-2939Crossref PubMed Scopus (24) Google Scholar). Opposite modifications of circulating levels of leptin and its soluble receptor also occur across the eating-disorder spectrum, with plasma levels of soluble leptin receptor significantly increased in patients with anorexia nervosa or bulimia nervosa, but decreased in patients with binge-eating disorder or those who are obese but are non-binge-eating (42Monteleone P. Fabrazzo M. Tortorella A. Fuschino A. Maj M. Mol. Psychiatry. 2002; 7: 641-646Crossref PubMed Scopus (63) Google Scholar). Soluble leptin receptor levels are also regulated by gender, adiposity, hormones, and rhLeptin administration (43Chan J.L. Bluher S. Yiannakouris N. Suchard M.A. Kratzsch J. Mantzoros C.S. Diabetes. 2002; 51: 2105-2112Crossref PubMed Scopus (207) Google Scholar). In aggregate, these studies suggest that the soluble leptin receptor may have important implications for the biological activity of leptin. Preliminary results of ongoing work in our laboratory using transgenic mice overexpressing the soluble leptin receptor support the hypothesis that increased soluble leptin receptor in wild-type mice is associated with decreased adiposity as well as decreased body weight. It is perceivable that future studies will shed more light on the usefulness of developing methods to regulate the generation of the soluble leptin receptor for therapeutic benefit. Leptin is an adipocyte-derived hormone of 167 amino acids (1Zhang Y. Proenca P. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11631) Google Scholar). It has potent weight-reducing effects in vivo (2Campfield L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3060) Google Scholar, 3Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4199) Google Scholar, 4Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3850) Google Scholar). Inob/ob mice, the gene encoding leptin is mutated, resulting in morbid obesity and associated abnormalities, including hyperphagia, hypothermia, diabetes, and infertility. The leptin receptor, OB-R, is a member of the cytokine receptor family (5Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3205) Google Scholar). It is encoded by the diabetes (db) gene, mutation of which also results in phenotypes similar to that exhibited byob/ob mice. OB-R is alternatively spliced into at least five transcripts from a single gene. These transcripts encode proteins that are called the long (OB-Rb), short (OB-Ra, -c, and -d), and soluble (OB-Re) forms of the leptin receptor. With the exception of the soluble leptin receptor, other receptor isoforms differ from each other by the alternative use of a unique terminal coding exon (6Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2095) Google Scholar). OB-Rb is essential in mediating leptin's weight-reducing effects via the hypothalamus (6Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2095) Google Scholar, 7Chen H. Charlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R.I. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1919) Google Scholar). OB-R is expressed in both the nervous system and in peripheral tissues. The relative expression levels of different receptor isoforms vary among tissues, possibly to allow leptin's biological activity to be more precisely regulated at various leptin target sites (8Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6231-6235Crossref PubMed Scopus (730) Google Scholar). OB-Rb is enriched in the hypothalamus, the site of leptin's action on food intake and body weight. Leptin activation of OB-Rb within this brain region results in the inhibition of neuropeptide Y/agouti-related protein neurons and activation of pro-opiomelanocortin neurons (9Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (626) Google Scholar). Leptin-activated pro-opiomelanocortin neurons become depolarized and release anorexigenic peptides; leptin-inhibited neuropeptide Y/agouti-related protein neurons become hyperpolarized and reduce the release of orexigenic peptides (10Cowley M.A. Smart J.L. Rubinstein M. Cerdan M.G. Diano S. Horvath T.L. Cone R.D. Low M.J. Nature. 2001; 411: 480-484Crossref PubMed Scopus (1754) Google Scholar). These neural circuits represent the main known downstream mediators of leptin's biological effect on food intake. OB-Rb can also activate signal transduction in a variety of peripheral tissues, including adipose tissue, T cells, endothelial cells, and pancreatic β-cells (11Kim Y.B. Uotani S. Pierroz D.D. Flier J.S. Kahn B.B. Endocrinology. 2000; 141: 2328-2339Crossref PubMed Scopus (196) Google Scholar, 12Lord G.M. Matarese G. Howard J.K. Baker R.J. Bloom S.R. Lechler R.I. Nature. 1998; 394: 897-901Crossref PubMed Scopus (1828) Google Scholar, 13Sierra-Honigmann M.R. Nath A.K. Murakami C. Garcia-Cardena G. Papapetropoulos A. Sessa W.C. Madge L.A. Schechner J.S. Schwabb M.B. Polverini P.J. Flores-Riveros J.R. Science. 1998; 281: 1683-1686Crossref PubMed Scopus (1266) Google Scholar, 14Unger R.H. Zhou Y.T. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2327-2332Crossref PubMed Scopus (372) Google Scholar, 15Huang L. Li C. Cell Res. 2000; 10: 81-92Crossref PubMed Scopus (187) Google Scholar). More recently, it is also demonstrated that leptin-induced fatty acid oxidation in muscle via 5′-AMP-activated protein kinase is mediated by both leptin acting on muscle directly and by functioning through the hypothalamic-sympathetic nervous system axis (16Minokoshi Y. Kim Y.B. Peroni O.D. Fryer L.G. Muller C. Carling D. Kahn B.B. Nature. 2002; 415: 339-343Crossref PubMed Scopus (1651) Google Scholar). Taken together, these results confirm that direct as well as indirect leptin signaling at these sites may be necessary for the many biological effects of leptin. In all cases, the presence of OB-Rb is required, asdb/db mice that are without this receptor isoform do not respond to leptin. Although OB-Rb is essential in mediating leptin's biological effects, other receptor isoforms may still be necessary for leptin to exert its full spectrum of in vivo functions. Among the short forms of the leptin receptor, OB-Ra is most abundantly expressed (17Fei H. Okano H.J. Li C. Lee G.-H. Zhao C. Darnell R. Friedman J.M. Prod. Natl. Acad. Sci., U. S. A. 1997; 94: 7001-7005Crossref PubMed Scopus (652) Google Scholar). It is enriched at the choroid plexus and brain microvessels, sites of the blood-cerebrospinal fluid barrier and the blood-brain barrier, suggesting that it may be involved in the transport of leptin across these barriers to reach the hypothalamus. Previously, we have shown that the secreted form of the leptin receptor, OB-Re, circulates in mouse plasma and is capable of binding to leptin (18Li C. Ioffe E. Fidahusein N. Connolly E. Friedman J.M. J. Biol. Chem. 1998; 273: 10078-10082Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In this report, we present evidence that circulating soluble leptin receptor may arise from both OB-Re mRNA and ectodomain shedding of membrane-spanning OB-R isoforms. We demonstrate that stable cell lines overexpressing OB-Ra or OB-Rb are capable of releasing soluble leptin receptor into the media by proteolytic cleavage, as detected by leptin-Sepharose pull-down assay. Furthermore, we demonstrate that adenovirus-mediated in vivooverexpression of OB-Ra or OB-Rb also results in release of soluble leptin receptor into plasma in mice via the same mechanism. Ectodomain shedding of both OB-Ra and OB-Rb is detected by a monoclonal antibody recognizing the FLAG epitope tag fused in-frame with the amino terminus of recombinant receptor protein. Stable 293 cells expressing OB-Ra and OB-Rb release the soluble leptin receptor under serum-free conditions, suggesting that the cleavage of membrane-bound receptor may be mediated by proteases resident in the cell. Because the soluble leptin receptor affects leptin's effect on food intake and body weight inob/ob mice (19Huang L. Wang Z. Li C. J. Biol. Chem. 2001; 276: 6343-6349Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), regulated ectodomain shedding of membrane-spanning leptin receptors may represent a novel mechanism of regulating leptin's biological activity. DISCUSSIONIn our earlier studies, we demonstrated that the soluble leptin receptor circulates in plasma and is capable of binding to leptin (18Li C. Ioffe E. Fidahusein N. Connolly E. Friedman J.M. J. Biol. Chem. 1998; 273: 10078-10082Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Our recently published results also demonstrated that the soluble receptor might play key roles in determining the amount of total leptin in circulation (19Huang L. Wang Z. Li C. J. Biol. Chem. 2001; 276: 6343-6349Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Other studies also suggest that expression and plasma concentration of the soluble leptin receptor may be regulated (29Gavrilova O. Barr V. Marcus-Samuels B. Reitman M. J. Biol. Chem. 1997; 272: 30546-30551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 30Widjaja A. Kielstein J.T. Horn R. von Zur Muhlen A. Kliem V. Brabant G. Nephrol. Dial. Transplant. 2000; 15: 846-850Crossref PubMed Scopus (41) Google Scholar). However, the origin of circulating soluble leptin receptor in plasma has not been clearly defined, especially as the mRNA splice variant encoding this receptor isoform in humans has not been found (26Chua Jr., S.C. Koutras I.K. Han L. Liu S.M. Kay J. Young S.J. Chung W.K. Leibel R.L. Genomics. 1997; 45: 264-270Crossref PubMed Scopus (125) Google Scholar).Because no post-translational modification of leptin occurs in vivo (31Cohen S.L. Halaas J.L. Friedman J.M. Chait B.T. Bennett L. Chang D. Hecht R. Collins F. Nature. 1996; 382: 589Crossref PubMed Scopus (91) Google Scholar), soluble leptin receptor may be an important factor regulating leptin's availability and bioactivity. This hypothesis is supported by findings that in human plasma, soluble leptin receptor represents the major leptin binding activity (27Lammert A. Kiess W. Bottner A. Glasow A. Kratzsch J. Biochem. Biophys. Res. Commun. 2001; 283: 982-988Crossref PubMed Scopus (243) Google Scholar).The main site of expression of the mRNA splice variant encoding the soluble leptin receptor in vivo remains unknown. Previously, we failed to detect a signal for OB-Re when Northern blot analysis was performed (17Fei H. Okano H.J. Li C. Lee G.-H. Zhao C. Darnell R. Friedman J.M. Prod. Natl. Acad. Sci., U. S. A. 1997; 94: 7001-7005Crossref PubMed Scopus (652) Google Scholar). Available data have demonstrated that in mice, OB-Re is expressed by the placenta. Its expression starts at day 14 of pregnancy, peaking just before parturition to about 40-fold the level found in non-pregnant mice (29Gavrilova O. Barr V. Marcus-Samuels B. Reitman M. J. Biol. Chem. 1997; 272: 30546-30551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 32Yamaguchi M. Murakami T. Yasui Y. Otani S. Kawai M. Kishi K. Kurachi H. Shima K. Aono T. Murata Y. Biochem. Biophys. Res. Commun. 1998; 252: 363-367Crossref PubMed Scopus (24) Google Scholar). In rats and humans, the pregnancy-associated rise of circulating leptin and its soluble receptor is relatively modest, achieving only a 2-fold increaseversus a more than 40-fold increase in mice (33Lewandowski K. Horn R. O'Callaghan C.J. Dunlop D. Medley G.F. O'Hare P. Brabant G. J. Clin. Endocrinol. Metab. 1999; 84: 300-306Crossref PubMed Scopus (130) Google Scholar).Our current study provided evidence of an additional mechanism for the generation of soluble leptin receptor by ectodomain shedding of membrane-spanning receptor, both in vitro and in vivo. This result is also in agreement with an earlier report on the generation of human soluble leptin receptor from transiently expressed membrane-spanning receptors in tissue culture (20Maamra M. Bidlingmaier M. Postel-Vinay M.C. Wu Z. Strasburger C.J. Ross R.J. Endocrinology. 2001; 142: 4389-4393Crossref PubMed Scopus (106) Google Scholar).Ectodomain shedding by proteolysis to yield soluble intercellular regulators has been observed for many proteins, such as tumor necrosis factor α and transforming growth factor α (34Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1356) Google Scholar). The responsible protease for these proteins, tumor necrosis factor α-converting enzyme, is a member of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinases. Molecules that are capable of inhibiting metalloproteinases are being tested as promising reagents for cancer therapy (35Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2349) Google Scholar). At the present time, the modulation of the soluble leptin receptor on leptin biology is not well understood. Initial evidence of the presence of free and bound leptin in human circulation was obtained by gel-filtration chromatography of plasma containing added 125I-leptin tracers (36Sinha M.K. Opentanova I. Ohannesian J.P. Kolaczynski J.W. Heiman M.L. Hale J. Becker G.W. Bowsher R.R. Stephens T.W. Caro J.F. J. Clin. Invest. 1996; 98: 1277-1282Crossref PubMed Scopus (515) Google Scholar). This and later studies showed that obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans, whereas weight loss increases it (37Laimer M. Ebenbichler C.F. Kaser S. Sandhofer A. Weiss H. Nehoda H. Aigner F. Patsch J.R. Obes. Res. 2002; 10: 597-601Crossref PubMed Scopus (74) Google Scholar, 38van Dielen F.M. van 't Veer C. Buurman W.A. Greve J.W. J. Clin. Endocrinol. Metab. 2002; 87: 1708-1716Crossref PubMed Scopus (117) Google Scholar, 39Lahlou N. Issad T. Lebouc Y. Carel J.C. Camoin L. Roger M. Girard J. Diabetes. 2002; 51: 1980-1985Crossref PubMed Scopus (35) Google Scholar, 40Ogier V. Ziegler O. Mejean L. Nicolas J.P. Stricker-Krongrad A. Int. J. Obes. Relat. Metab. Disord. 2002; 26: 496-503Crossref PubMed Scopus (71) Google Scholar). In others studies, obese and normal weight individuals were found to have similar amount of total circulating soluble leptin receptor, yet in the obese most soluble leptin receptor is bound to leptin, whereas in lean individuals a much smaller percentage of soluble leptin receptor is bound to leptin (75% in the obese versus 33% in the lean); thus, the inability to up-regulate circulating soluble leptin receptor could be a factor in the pathogenesis of obesity (36Sinha M.K. Opentanova I. Ohannesian J.P. Kolaczynski J.W. Heiman M.L. Hale J. Becker G.W. Bowsher R.R. Stephens T.W. Caro J.F. J. Clin. Invest. 1996; 98: 1277-1282Crossref PubMed Scopus (515) Google Scholar, 38van Dielen F.M. van 't Veer C. Buurman W.A. Greve J.W. J. Clin. Endocrinol. Metab. 2002; 87: 1708-1716Crossref PubMed Scopus (117) Google Scholar, 41Wu Z. Bidlingmaier M. Liu C. De Souza E.B. Tschop M. Morrison K.M. Strasburger C.J. J. Clin. Endocrinol. Metab. 2002; 87: 2931-2939Crossref PubMed Scopus (24) Google Scholar). However, a larger percentage of leptin circulates in the free form in the obese (41Wu Z. Bidlingmaier M. Liu C. De Souza E.B. Tschop M. Morrison K.M. Strasburger C.J. J. Clin. Endocrinol. Metab. 2002; 87: 2931-2939Crossref PubMed Scopus (24) Google Scholar). Opposite modifications of circulating levels of leptin and its soluble receptor also occur across the eating-disorder spectrum, with plasma levels of soluble leptin receptor significantly increased in patients with anorexia nervosa or bulimia nervosa, but decreased in patients with binge-eating disorder or those who are obese but are non-binge-eating (42Monteleone P. Fabrazzo M. Tortorella A. Fuschino A. Maj M. Mol. Psychiatry. 2002; 7: 641-646Crossref PubMed Scopus (63) Google Scholar). Soluble leptin receptor levels are also regulated by gender, adiposity, hormones, and rhLeptin administration (43Chan J.L. Bluher S. Yiannakouris N. Suchard M.A. Kratzsch J. Mantzoros C.S. Diabetes. 2002; 51: 2105-2112Crossref PubMed Scopus (207) Google Scholar). In aggregate, these studies suggest that the soluble leptin receptor may have important implications for the biological activity of leptin. Preliminary results of ongoing work in our laboratory using transgenic mice overexpressing the soluble leptin receptor support the hypothesis that increased soluble leptin receptor in wild-type mice is associated with decreased adiposity as well as decreased body weight. It is perceivable that future studies will shed more light on the usefulness of developing methods to regulate the generation of the soluble leptin receptor for therapeutic benefit. In our earlier studies, we demonstrated that the soluble leptin receptor circulates in plasma and is capable of binding to leptin (18Li C. Ioffe E. Fidahusein N. Connolly E. Friedman J.M. J. Biol. Chem. 1998; 273: 10078-10082Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Our recently published results also demonstrated that the soluble receptor might play key roles in determining the amount of total leptin in circulation (19Huang L. Wang Z. Li C. J. Biol. Chem. 2001; 276: 6343-6349Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Other studies also suggest that expression and plasma concentration of the soluble leptin receptor may be regulated (29Gavrilova O. Barr V. Marcus-Samuels B. Reitman M. J. Biol. Chem. 1997; 272: 30546-30551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 30Widjaja A. Kielstein J.T. Horn R. von Zur Muhlen A. Kliem V. Brabant G. Nephrol. Dial. Transplant. 2000; 15: 846-850Crossref PubMed Scopus (41) Google Scholar). However, the origin of circulating soluble leptin receptor in plasma has not been clearly defined, especially as the mRNA splice variant encoding this receptor isoform in humans has not been found (26Chua Jr., S.C. Koutras I.K. Han L. Liu S.M. Kay J. Young S.J. Chung W.K. Leibel R.L. Genomics. 1997; 45: 264-270Crossref PubMed Scopus (125) Google Scholar). Because no post-translational modification of leptin occurs in vivo (31Cohen S.L. Halaas J.L. Friedman J.M. Chait B.T. Bennett L. Chang D. Hecht R. Collins F. Nature. 1996; 382: 589Crossref PubMed Scopus (91) Google Scholar), soluble leptin receptor may be an important factor regulating leptin's availability and bioactivity. This hypothesis is supported by findings that in human plasma, soluble leptin receptor represents the major leptin binding activity (27Lammert A. Kiess W. Bottner A. Glasow A. Kratzsch J. Biochem. Biophys. Res. Commun. 2001; 283: 982-988Crossref PubMed Scopus (243) Google Scholar). The main site of expression of the mRNA splice variant encoding the soluble leptin receptor in vivo remains unknown. Previously, we failed to detect a signal for OB-Re when Northern blot analysis was performed (17Fei H. Okano H.J. Li C. Lee G.-H. Zhao C. Darnell R. Friedman J.M. Prod. Natl. Acad. Sci., U. S. A. 1997; 94: 7001-7005Crossref PubMed Scopus (652) Google Scholar). Available data have demonstrated that in mice, OB-Re is expressed by the placenta. Its expression starts at day 14 of pregnancy, peaking just before parturition to about 40-fold the level found in non-pregnant mice (29Gavrilova O. Barr V. Marcus-Samuels B. Reitman M. J. Biol. Chem. 1997; 272: 30546-30551Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 32Yamaguchi M. Murakami T. Yasui Y. Otani S. Kawai M. Kishi K. Kurachi H. Shima K. Aono T. Murata Y. Biochem. Biophys. Res. Commun. 1998; 252: 363-367Crossref PubMed Scopus (24) Google Scholar). In rats and humans, the pregnancy-associated rise of circulating leptin and its soluble receptor is relatively modest, achieving only a 2-fold increaseversus a more than 40-fold increase in mice (33Lewandowski K. Horn R. O'Callaghan C.J. Dunlop D. Medley G.F. O'Hare P. Brabant G. J. Clin. Endocrinol. Metab. 1999; 84: 300-306Crossref PubMed Scopus (130) Google Scholar). Our current study provided evidence of an additional mechanism for the generation of soluble leptin receptor by ectodomain shedding of membrane-spanning receptor, both in vitro and in vivo. This result is also in agreement with an earlier report on the generation of human soluble leptin receptor from transiently expressed membrane-spanning receptors in tissue culture (20Maamra M. Bidlingmaier M. Postel-Vinay M.C. Wu Z. Strasburger C.J. Ross R.J. Endocrinology. 2001; 142: 4389-4393Crossref PubMed Scopus (106) Google Scholar). Ectodomain shedding by proteolysis to yield soluble intercellular regulators has been observed for many proteins, such as tumor necrosis factor α and transforming growth factor α (34Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1356) Google Scholar). The responsible protease for these proteins, tumor necrosis factor α-converting enzyme, is a member of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinases. Molecules that are capable of inhibiting metalloproteinases are being tested as promising reagents for cancer therapy (35Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2349) Google Scholar). At the present time, the modulation of the soluble leptin receptor on leptin biology is not well understood. Initial evidence of the presence of free and bound leptin in human circulation was obtained by gel-filtration chromatography of plasma containing added 125I-leptin tracers (36Sinha M.K. Opentanova I. Ohannesian J.P. Kolaczynski J.W. Heiman M.L. Hale J. Becker G.W. Bowsher R.R. Stephens T.W. Caro J.F. J. Clin. Invest. 1996; 98: 1277-1282Crossref PubMed Scopus (515) Google Scholar). This and later studies showed that obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans, whereas weight loss increases it (37Laimer M. Ebenbichler C.F. Kaser S. Sandhofer A. Weiss H. Nehoda H. Aigner F. Patsch J.R. Obes. Res. 2002; 10: 597-601Crossref PubMed Scopus (74) Google Scholar, 38van Dielen F.M. van 't Veer C. Buurman W.A. Greve J.W. J. Clin. Endocrinol. Metab. 2002; 87: 1708-1716Crossref PubMed Scopus (117) Google Scholar, 39Lahlou N. Issad T. Lebouc Y. Carel J.C. Camoin L. Roger M. Girard J. Diabetes. 2002; 51: 1980-1985Crossref PubMed Scopus (35) Google Scholar, 40Ogier V. Ziegler O. Mejean L. Nicolas J.P. Stricker-Krongrad A. Int. J. Obes. Relat. Metab. Disord. 2002; 26: 496-503Crossref PubMed Scopus (71) Google Scholar). In others studies, obese and normal weight individuals were found to have similar amount of total circulating soluble leptin receptor, yet in the obese most soluble leptin receptor is bound to leptin, whereas in lean individuals a much smaller percentage of soluble leptin receptor is bound to leptin (75% in the obese versus 33% in the lean); thus, the inability to up-regulate circulating soluble leptin receptor could be a factor in the pathogenesis of obesity (36Sinha M.K. Opentanova I. Ohannesian J.P. Kolaczynski J.W. Heiman M.L. Hale J. Becker G.W. Bowsher R.R. Stephens T.W. Caro J.F. J. Clin. Invest. 1996; 98: 1277-1282Crossref PubMed Scopus (515) Google Scholar, 38van Dielen F.M. van 't Veer C. Buurman W.A. Greve J.W. J. Clin. Endocrinol. Metab. 2002; 87: 1708-1716Crossref PubMed Scopus (117) Google Scholar, 41Wu Z. Bidlingmaier M. Liu C. De Souza E.B. Tschop M. Morrison K.M. Strasburger C.J. J. Clin. Endocrinol. Metab. 2002; 87: 2931-2939Crossref PubMed Scopus (24) Google Scholar). However, a larger percentage of leptin circulates in the free form in the obese (41Wu Z. Bidlingmaier M. Liu C. De Souza E.B. Tschop M. Morrison K.M. Strasburger C.J. J. Clin. Endocrinol. Metab. 2002; 87: 2931-2939Crossref PubMed Scopus (24) Google Scholar). Opposite modifications of circulating levels of leptin and its soluble receptor also occur across the eating-disorder spectrum, with plasma levels of soluble leptin receptor significantly increased in patients with anorexia nervosa or bulimia nervosa, but decreased in patients with binge-eating disorder or those who are obese but are non-binge-eating (42Monteleone P. Fabrazzo M. Tortorella A. Fuschino A. Maj M. Mol. Psychiatry. 2002; 7: 641-646Crossref PubMed Scopus (63) Google Scholar). Soluble leptin receptor levels are also regulated by gender, adiposity, hormones, and rhLeptin administration (43Chan J.L. Bluher S. Yiannakouris N. Suchard M.A. Kratzsch J. Mantzoros C.S. Diabetes. 2002; 51: 2105-2112Crossref PubMed Scopus (207) Google Scholar). In aggregate, these studies suggest that the soluble leptin receptor may have important implications for the biological activity of leptin. Preliminary results of ongoing work in our laboratory using transgenic mice overexpressing the soluble leptin receptor support the hypothesis that increased soluble leptin receptor in wild-type mice is associated with decreased adiposity as well as decreased body weight. It is perceivable that future studies will shed more light on the usefulness of developing methods to regulate the generation of the soluble leptin receptor for therapeutic benefit. We thank Dr. J. Darnell (Rockefeller University) for kindly providing us the STAT3 luciferase reporter construct, Drs. T.-C. He and B. Vogelstein (Johns Hopkins University) for pAdEasy vectors to generate adenoviruses, and Drs. R. H. Unger, C. B. Newgard, and G. Yang for insightful discussions and/or a critical reading of the manuscript.

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