Tools Now Exist to Measure Circulating IGF-Binding Protein-3 Proteolysis, to Study Its Regulation and Its Effects on IGF-I Bioavailability

Michel Binoux

Institut National de la Santé et de la Recherche Médicale, Unité 515 Hôpital Saint Antoine Paris, France

Address correspondence to: Michel Binoux, M.D., INSERM Unité 515, Hôpital Saint Antoine, 75571 Paris, France.

To the editor:

A long-standing debate is revived by Robert Baxter’s letter to the editor concerning the physiological significance of IGF-binding protein-3 (IGFBP-3) proteolysis in the circulation. Although debate continues, the letter provides no new arguments. It is somewhat surprising, however, that it was sparked by a purely methodological publication (1)—unless the author feels that the rationale for instigating such research was groundless until definitive experimental proof has been provided that IGFBP-3 proteolysis can increase the bioavailability of IGFs to its target tissues, although, in his words, "indeed, the definitive experiment is difficult to envisage."

The quotes selected from the introduction to our article as the basis for the criticism allude to earlier work that needs to be restored to its rightful scientific context. "The authors ... point out that in human pregnancy virtually all circulating IGFBP is degraded ..." The term "degraded" naturally refers to "limited proteolysis," with which the cited sentence begins. It has been clearly demonstrated that in pregnancy serum the characteristic 42-39-kDa doublet corresponding to IGFBP-3 is no longer detectable in Western ligand blotting, indicating functional alteration (2, 3). Western immunoblot analysis confirms this disappearance of the intact IGFBP-3 doublet, with concomitant increase of an approximately 30-kDa fragment that is consistently detected in serum, sometimes accompanied by 20- and 16-kDa fragments. Its proportional quantities relative to the 42-39-kDa form vary with physiopathological status, which strongly points toward physiological significance (4, 5, 6).

"The authors state that proteolysed IGFBP-3 loses affinity for IGFs which are then redistributed towards the 40-kDa binary IGFBP-IGF complexes." This quote omits the end of the sentence, which was "... and the free fraction of IGFs." The sentence summarized the conclusions of an extensive study previously undertaken (7), which merits reiteration, since Baxter chooses to cite publications with opposing conclusions. In that study, it was clearly shown that in pregnancy plasma, free IGF-I is enriched at the expense of the fraction in the 140-kDa complexes, whereas IGF-II is concentrated in the 140-kDa complexes at the expense of the 40-kDa complexes. The redistribution of the IGFs among the three circulating pools results from the loss of affinity of IGFBP-3 for IGFs, particularly IGF-I, and their accelerated kinetics of dissociation at 37 C. The results of a subsequent study by Lee and Rechler (8) supported this interpretation. In addition, Davenport et al. (9) demonstrated that 125I-IGF-I injected into pregnant rats was cleared from the serum five times faster than that injected into nonpregnant females. Urinary clearance was not significantly increased and 125I-IGF-I did not cross the placenta. They concluded that tissue uptake of IGF-I is increased in the mother in response to the metabolic needs of gestation. (9) The potential implications of IGFBP-3 proteolysis in IGF bioavailability have further been demonstrated by the more marked biological effects of pregnancy serum than normal serum in a chick embryo fibroblast assay (10).

The results of studies based on assays of the "free" fraction of IGF in serum are also germane. Relative to total IGF-I, this fraction is approximately doubled in pregnancy (11). Also, treatment of breast cancer patients with the anti-tumor drug suramin induces near-complete proteolysis of serum IGFBP-3 associated with an elevation in levels of "free" IGF-I (12). Interestingly, Baxter maintains that "the biological significance of these measurements is unclear."

It is possible that acid or SDS treatment provokes "further damage to the proteolysed protein" and amplifies the loss of affinity for IGF-I. Baxter and Skriver (13) have shown that this loss is even greater for 125I-IGF-I, possibly through alteration of tyrosine residues 24 and 60 during the iodination process (13). Even so, far from being a drawback, this is advantageous in our ligand immunofunctional assay where the aim is specifically to detect intact IGFBP-3 and exclude its proteolytic fragments.

We do not consider it speculative to state that "the LIFA for IGFBP-3 opens new perspectives in investigating the regulation of IGFBP-3 proteolysis and IGF-I bioavailability." First, the method furnishes a sensitivity, precision, and reproducibility of quantification of intact IGFBP-3 beyond the capabilities of the semiquantitative examination of immunoblots. Secondly, associated with the classical RIAs of IGFBP-3 and IGF-I, it allows of quantification of IGFBP-3 proteolysis and use of the IGF-I/intact IGFBP-3 ratio as an index of the exchangeable (bioavailable) fraction of IGFBP-3-bound IGF-I. The first practical applications of the technique were published in the May issue of this journal (14). Apart from classical indications in clinical investigation in cases of abnormal GH secretion and growth and nutritional disorders, determination of the proportions of intact and proteolysed IGFBP-3 as related to IGF-I levels will be useful, not least in view of epidemiological studies suggesting a link between high plasma IGF-I and low IGFBP-3 levels and the risk of prostate, breast, and colorectal cancers (15). Different approaches from ours are certainly valid for quantifying the intact and proteolysed forms of IGFBP-3. This is so in the novel ELISAs capable of differential determination of intact and fragmented IGFBP-3 variants developed by Diagnostic Systems Laboratories, Inc. (16).

Received May 24, 2001.

References

  1. Lassarre C, Binoux M 2001 Measurement of intact insulin-like growth factor binding protein-3 in human plasma using a ligand immunofunctional assay. J Clin Endocrinol Metab 86:1260–1266[Abstract/Free Full Text]
  2. Hossenlopp P, Segovia B, Lassarre C, Roghani M, Bredon M, Binoux M 1990 Evidence of enzymatic degradation of insulin-like growth factor binding proteins in the "150 K" complex during pregnancy. J Clin Endocrinol Metab 71:797–805[Abstract]
  3. Giudice LC, Farrell EM, Pham H, Lamson G, Rosenfeld RG 1990 Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: effects of a pregnancy-associated serum protease activity. J Clin Endocrinol Metab 71:806–816[Abstract]
  4. Gargosky SE, Pham HM, Wilson KF, Liu F, Guidice LC, Rosenfeld RG 1992 Measurement and characterization of insulin-like growth factor binding protein-3 in human biological fluids: discrepencies between radioimmunoassay and ligand blotting. Endocrinology 131:3051–3060[Abstract]
  5. Lalou C, Binoux M 1993 Evidence that limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) occurs in the normal state outside of the bloodstream. Regul Pept 48:179–188[CrossRef][Medline]
  6. Maile L, Brown AJ, Holly JMP 1999 IGF binding protein proteolysis in various clinical states. In: Rosenfeld RG, Roberts Jr CT, eds. The IGF system: molecular biology, physiology and clinical applications. Totowa, NJ: Humana Press Inc.; 633–649
  7. Lassarre C, Binoux M 1994 Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma. Endocrinology 134:1254–1262[Abstract]
  8. Lee CY, Rechler MM 1996 Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in 150-kilodalton IGFBP complexes by a cation-dependent protease activity in adult rat serum promotes the release of bound IGF-I. Endocrinology 137:2051–2058[Abstract]
  9. Davenport ML, Clemmons DR, Miles MV, Camacho-Hübner C, D’Ercole AJ, Underwood LE 1990 Regulation of serum insulin-like growth factor-I (IGF-I) and IGF binding proteins during rat pregnancy. Endocrinology 127:1278–1286[Abstract]
  10. Blat C, Villaudy J, Binoux M 1994 In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. J Clin Invest 93:2286–2290[Medline]
  11. Hasegawa T, Hasegawa Y, Takada M, Tsuchiya Y 1995 The free form of insulin-like growth factor I increases in circulation during normal human pregnancy. J Clin Endocrinol Metab 80:3284–3286[Abstract]
  12. Lawrence JB, Conover CA, Haddad TC, et al. 1997 Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-like growth factors (IGFs) and IGF binding proteins. Clin Cancer Res 3:1713–1720[Abstract]
  13. Baxter RC, Skriver L 1993 Altered ligand specificity of proteolysed insulin-like growth factor binding protein-3. Biochem Biophys Res Commun 198:1267–1273[CrossRef]
  14. Lassarre C, Duron F, Binoux M 2001 Use of the ligand immunofunctional assay for human insulin-like growth factor binding protein-3 (IGFBP-3) to analyse IGFBP-3 proteolysis and IGF-I bioavailability in healthy adults, GH-deficient and acromegalic patients, and diabetics. J Clin Endocrinol Metab 86:1942–1952[Abstract/Free Full Text]
  15. Giovannucci E 1999 Insulin-like growth factor I and binding protein-3 and risk of cancer. Horm Res 51 (Suppl 3):34–41
  16. Diamandi A, Mistry J, Krishna RG, Khosravi J 2000 Immunoassay of insulin-like growth factor-binding protein-3 (IGFBP-3): new means to quantifying IGFBP-3 proteolysis. J Clin Endocrinol Metab 85:2327–2333[Abstract/Free Full Text]




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