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Original Article |
Endocrine Research Unit, Mayo Clinic and Mayo Foundation (S.K., C.A.C.), Rochester, Minnesota 55905; and GroPep Ltd. (J.B.), Thebarton, South Australia 5031, Australia
Address all correspondence and requests for reprints to: Sundeep Khosla, M.D., Mayo Clinic, 200 First Street SW, 5-194 Joseph, Rochester, Minnesota 55905. E-mail: . khosla.sundeep{at}mayo.edu
Abstract
Hepatitis C-associated osteosclerosis (HCAO) is a rare syndrome of adult-onset osteosclerosis. An understanding of the factor(s) leading to the stimulation of bone formation in these patients may provide novel anabolic approaches for the treatment of osteoporosis. We have demonstrated that HCAO patients have a specific increase in circulating big IGF-II (IGF-IIE) and IGF-binding protein-2 (IGFBP-2) levels, and that IGF-IIE and IGFBP-2 circulate together in a bioavailable, 50-kDa complex. Patients with nonislet cell tumor hypoglycemia (NICTH) also have increased circulating IGF-IIE and IGFBP-2 levels. However, HCAO patients do not exhibit hypoglycemia, nor do NICTH patients exhibit obvious osteosclerosis. Thus, to better understand the reason(s) for the differing clinical manifestations of the IGF-IIE excess in the two syndromes, we characterized IGF-IIE in HCAO and NICTH sera using recently developed antibodies (Ab) recognizing either the full-length IGF-IIE 89-amino acid C-terminal extension peptide (IIE138–156 Ab) or specific cleavage forms of IGF-IIE (IIE78–88 Ab and IIE89–101 Ab). The predominant IGF-IIE form in HCAO serum migrated on SDS-PAGE as a single band at approximately 18 kDa that reacted with the IIE89–101 Ab. On the other hand, the predominant form in NICTH serum migrated as a doublet of 14 and 16 kDa that reacted with the IIE78–88 Ab. There results are consistent with differential processing of the IGF-IIE precursor at predicted cleavage sites producing IGF-IIE1–104 and IGF-IIE1–88 in HCAO and NICTH, respectively. As these two forms may have differing biological activities and/or targeting properties, our findings may explain at least in part the different manifestations of IGF-IIE overproduction in the two syndromes.
HEPATITIS C-associated osteosclerosis (HCAO) is a rare syndrome of adult-onset osteosclerosis (1, 2, 3, 4, 5, 6, 7). These patients have remarkable increases in their bone mass consequent to a marked stimulation of bone formation. Despite its rare occurrence, HCAO is of great interest, because an understanding of the factor(s) leading to the stimulation of bone formation in these patients may provide novel approaches for the treatment of osteoporosis.
We have previously demonstrated that HCAO patients have a specific increase in circulating big IGF-II (IGF-IIE) and IGFBP-2 levels, and that IGF-IIE and IGFBP-2 circulate together in a bioavailable, approximately 50-kDa complex (8). On the basis of these findings and in vitro studies demonstrating high avidity of the IGF-II/IGFBP-2 complex for human osteoblast extracellular matrix, we postulated that IGFBP-2 was targeting IGF-IIE to the skeleton in these patients, resulting in the stimulation of bone formation (8).
Similar to HCAO patients, patients with nonislet cell tumor hypoglycemia (NICTH) have increased circulating IGF-IIE and IGFBP-2 levels (9). However, HCAO patients do not exhibit hypoglycemia, nor have NICTH patients been reported to have osteosclerosis. As IGF-II is synthesized as a 156 amino acid precursor with potential cleavage sites after amino acid 67 (leading to mature IGF-II) as well as after amino acids 88 and 104 (leading to E-domain extensions of 21 and 37 amino acids, respectively) (10) (Fig. 1
), we postulated that the differing clinical manifestations of IGF-IIE excess in HCAO vs. NICTH may be due at least in part to differential processing of the IGF-II prohormone in the two syndromes. To directly test this hypothesis, we generated specific antisera directed against the 78–88, 89–101, or 138–156 regions of the E domain of IGF-IIE and examined HCAO vs. NICTH serum for immunoreactivity against each of these antisera. Our results indicate that the predominant circulating forms of IGF-IIE in HCAO and NICTH are clearly different, perhaps accounting for the development of osteosclerosis in one syndrome and hypoglycemia in the other.
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Generation of IGF-IIE region-specific Ab
Polyclonal Ab recognizing either the full-length IGF-IIE 89-amino acid C-terminal extension peptide [IIE138–156 antibody (Ab)] or specific cleavage forms of IGF-IIE (IIE78–88 Ab and IIE89–101 Ab; Fig. 1
) were developed. Synthetic peptides corresponding to regions of the E domain of IGF-II were synthesized and conjugated to diphtheria toxin using a maleimidocapryol-N-hydroxysuccinimide linker (Chiron Corp., Clayton, Australia). The sequences of these peptides are as follows: IGF-IIE78–88, NH2-Pro-Asp-Asn-Phe-Pro-Arg-Tyr-Pro-Val-Gly-Lys-COOH; IGF-IIE89–101, NH2-Phe-Phe-Gln-Tyr-Asp-Thr-Trp-Lys-Gln-Ser-Thr-Gln- Arg-COOH; and IGF-IIE138–156, NH2-Phe-Thr-Gln-Asp-Pro-Ala-His-Gly-Gly-Ala-Pro-Pro-Glu-Met-Ala-Ser-Asn-Arg-Lys-COOH. New Zealand White rabbits were immunized with the above peptide conjugates by repeated sc injection with Freund’s adjuvant, and immune sera were collected according to standard techniques (11).
The reactivity of the IGF-IIE antisera with unconjugated peptides and also recombinant IGF-II1–67 and IGF-IIE1–156 (GroPep Ltd., Adelaide, Australia) was tested by enzyme immunoassay. Maxisorp 96 plates (Nunc, Naperville, IL) were coated with 50 ng peptide in PBS and then washed with PBS and 0.1% Tween 20 (PBS-T). The wells were then blocked with PBS-T and 0.5% BSA for 60 min at 37 C, washed twice with PBS-T, and incubated with primary Ab at 1:1000 in PBS-T for 120 min at 37 C. The wells were then washed with PBS-T, incubated with antirabbit horseradish peroxidase-linked secondary Ab in PBS-T, and finally washed with PBS-T. The secondary reagent was then detected with 1 mg/ml O-phenylene-diamine chloride and 0.1% hydrogen peroxide in a citrate-phosphate buffer at pH 5.5, the reaction was terminated with 1 M H2SO4, and the OD490 was determined.
Immunoblotting of IGF-II, IGF-IIE1–104, and IGF-IIE1–156
Three-microgram samples of each growth factor were run on 7.5–20% SDS-PAGE under reducing conditions (100 mM dithiothreitol), transferred to nitrocellulose, and then stained with Ponceau Red. The growth factors used in these studies were produced using recombinant DNA techniques (GroPep Ltd.). The membranes were then cut into thirds to match the loading protocol (lanes 1–3, 4–6, and 7–9) before being developed with one of the three primary Ab. After blocking with either nonfat dry milk or BSA in Tris-buffered saline/0.1% Tween 20, the membranes were incubated overnight at 4 C in primary Ab, washed, and incubated in secondary Ab (goat antirabbit) for 1 h at room temperature. After subsequent washing, they were visualized by an enhanced chemiluminescent detection system (Amersham Biotech, Piscataway, NJ).
Immunoblotting of serum
After informed consent and approval of the local institutional review board were granted, serum was obtained from normal healthy subjects, two patients with HCAO who have been previously described (9), and patients with NICTH. The HCAO sera were obtained at a time when the patients still had elevated bone formation rates, as described in our previous publication (5). The NICTH sera were all obtained while the subjects still had their tumors. NICTH serum was provided by Dr. Raymond Hintz (Stanford University, Stanford, CA; one sample) and Dr. Naomi Hizuka (Tokyo Women’s Medical College, Tokyo, Japan; six samples). For the immunoblotting studies, a 1-µl serum sample was run on 7.5–20% SDS-PAGE under reducing conditions (100 mM dithiothreitol), transferred to nitrocellulose, and blocked with either nonfat dry milk or BSA in Tris-buffered saline/0.1% Tween 20. Membranes were incubated in primary and secondary Ab, and visualization systems were as described above.
Results
Immune sera directed against each of the epitopes in the E domain of IGF-IIE were generated as described in Materials and Methods. The specificities of these antisera were determined by enzyme immunoassay and are summarized in Table 1. The three antisera did not react with mature IGF-II, but did react with a recombinant IGF-IIE corresponding to the full-length 1–156 amino acid IGF-II precursor. As expected, the three antisera were also reactive with their cognate peptides within the E domain, but did not react with the other peptides. The ability of the three antisera to react with IGF-IIE was also confirmed by Western blot analysis with the mature 67-amino acid IGF-II, a 104-amino acid form of IGF-IIE, and a 156-amino acid form (Fig. 2). It is evident that the IGF-IIE preparations visualized with Ponceau Red staining are not single bands, but migrate as several forms, presumably due to partial glycosylation (Fig. 2A). As expected, subsequent Western blotting with the different Ab (Fig. 2B) demonstrated that the IIE78–88 and IIE89–101 antisera detected both of the IGF-IIE forms, but not mature IGF-II. The IIE138–156 antiserum only detected IGF-IIE1–156 again as expected.
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). Thus, the IIE78–88 Ab detected strong bands in the NICTH serum at approximately 14–16 kDa. A weak signal corresponding to the lower of the two bands in NICTH serum was also present in normal and HCAO sera. In addition, the IIE78–88 Ab weakly detected a higher molecular mass band at approximately 18 kDa in HCAO serum. This higher molecular mass band in HCAO serum reacted strongly with the IIE89–101 Ab. The IIE138–156 Ab detected a weak band at an even higher molecular mass in HCAO serum as well as some lower molecular mass bands in all three sera, which may represent nonspecific binding by this Ab.
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shows data from six additional NICTH sera and the HCAO serum used in Fig. 3 as well as an additional HCAO sample using the IIE78–88 Ab. As shown, there were multiple bands in the NICTH samples at 14–16 kDa. These were present to a much lesser extent in normal or HCAO serum, although the latter again had the higher molecular mass form of approximately 18 kDa.
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. Again, HCAO serum reacted strongly with this Ab, giving a band of approximately 18 kDa. This was not present to any significant extent in NICTH serum.
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We report the generation and characterization of Ab recognizing various cleavage forms of IGF-IIE and the use of these Ab in defining possible differences in the circulating forms of IGF-IIE in HCAO and NICTH. Based on the known cleavage sites for IGF-IIE (10), these Ab are capable of specifically recognizing either IGF-IIE1–88, IGF-IIE1–104, or full-length IGF-IIE1–156.
Our characterization of HCAO vs. NICTH serum demonstrates that the predominant form of IGF-IIE in HCAO serum migrated on SDS-PAGE as a single band at approximately 18 kDa and reacted with the IIE89–101 Ab. On the other hand, the predominant form in NICTH serum migrated as a doublet of approximately 14 and 16 kDa and reacted with the IIE78–88 Ab. These results are consistent with differential processing of the IGF-IIE precursor at predicted cleavage sites producing IGF-IIE1–104 and IGF-IIE1–88 in HCAO and NICTH, respectively. Our findings using these specific Ab are consistent with previous studies attempting to identify the form of IGF-IIE that is elevated in patients with NICTH. Thus, Hizuka et al. (12) performed Western analysis of serum from 10 patients with NICTH using an anti-IGF-II Ab and found that most of the IGF-II immunoreactivity migrated between 11 and 18 kDa. This was reduced in size to approximately 9.5 kDa after neuraminidase and O-glycosidase digestion, consistent with the predominant circulating form of IGF-IIE in NICTH sera being IGF-IIE1–88 (12). We also found that the IIE78–88 Ab detected multiple bands in NICTH sera, presumably corresponding to variable glycosylation of the IGF-IIE. Finally, the IIE138–156 Ab failed to recognize a specific band in either HCAO or NICTH sera, indicating that intact IGF-IIE1–156 is not present in any significant amount in the circulation in either of these conditions. We did attempt to deglycosylate the IGF-IIE in HCAO serum using neuraminidase and O-glycanase, but were unable to show any clear changes in the size of the observed band (data not shown). This would suggest that either the IGF-IIE in HCAO is not glycosylated (which appears to be an unlikely possibility), or that for some reason this peptide in HCAO serum is extremely resistant to standard deglycosylation methods.
A clear limitation of this study is the relatively small number of HCAO (n = 2) and NICTH (n = 7) subjects analyzed, because both are rare syndromes, and samples are difficult to obtain. Nonetheless, as noted earlier, our findings in the NICTH patients are entirely consistent with the apparent size of IGF-IIE previously identified in these patients (12) attesting (to some degree) to the robustness of the data. In addition, the major finding of our study has to do with qualitative differences (i.e. clearly different patterns of immunoreactivity with site-specific Ab) in IGF-IIE in HCAO vs. NICTH, rather than quantitative differences in IGF-IIE levels in the two syndromes, making issues of sample size somewhat less of a concern. Nonetheless, we recognize this limitation, and additional studies characterizing the IGF-IIE peptide in a larger number of HCAO and NICTH sera are clearly warranted to further validate our findings.
As the IGF-IIE1–104 and IGF-IIE1–88 isoforms may have differing biological activities and/or targeting properties, our findings may explain at least in part the different clinical manifestations of IGF-IIE overproduction in HCAO vs. NICTH. In particular, a major abnormality in NICTH, due perhaps directly to the excess IGF-IIE in the serum of these patients, is the disruption of the approximately 150-kDa ternary complex with a consequent shift of the IGFs and IGFBP-3 to the approximately 50-kDa binary complex and an increase in free IGFs, with resultant hypoglycemia (9). By contrast, we have previously shown that the ternary complex remains intact in HCAO serum, and as such, there is no increase in free IGFs in the circulation of these patients (8), probably explaining the absence of hypoglycemia in HCAO patients. Whether the disruption of the ternary complex in NICTH and its preservation in HCAO are due to the different forms of IGF-IIE present in the circulation of these patients is an issue that requires further study.
We have also previously found that the IGF-IIE in HCAO patients circulates bound to IGFBP-2 in the approximately 50-kDa binary fraction (8). Moreover, our previous in vitro studies indicate that upon binding IGF-II, IGFBP-2 has greatly enhanced avidity for the osteoblast extracellular matrix (8). Thus, we have postulated that the IGF-IIE/IGFBP-2 complex accumulates in bone in HCAO patients, and the subsequent local release of IGF-IIE in the bone microenvironment results in the stimulation of bone formation and osteosclerosis observed in these patients. Further studies more directly testing this hypothesis are currently underway. Indeed, based on the findings of the present study, IGF-IIE1–104 appears to be the IGF-IIE fragment that should be examined in combination with IGFBP-2 in these studies, because this the IGF-IIE isoform that is elevated in vivo in the HCAO patients.
In summary, we describe the generation and characterization of Ab specific for various potential cleavage forms of IGF-IIE. These Ab have provided powerful tools to characterize the forms of IGF-IIE that are elevated in HCAO vs. NICTH serum. The insights from these studies may help both in explaining the differing clinical manifestations of the IGF-IIE excess in the two syndromes as well as in selecting the optimal form of IGF-IIE to use in future studies aimed at developing novel anabolic approaches to treating osteoporosis.
Acknowledgments
We thank Laurie Bale, Phillip Elliott, and Leanne McGrath for technical assistance. We also thank Drs. Raymond Hintz and Naomi Hizuka for providing NICTH sera.
Footnotes
This work was supported by NIA Grant AG-04875. This paper was presented in part at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000.
Abbreviations: Ab, Antibodies; HCAO, hepatitis C-associated osteosclerosis; IGF-IIE, big IGF-II; IGFBP-2, IGF-binding protein-2; NICTH, nonislet cell tumor hypoglycemia; PBS-T, PBS and 0.1% Tween 20.
Received November 28, 2001.
Accepted May 9, 2002.
References
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