Stanniocalcin-2 inhibits mammalian growth by proteolytic inhibition of the insulin-like growth factor axis

Abstract

Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system.

Keywords: Insulin-like Growth Factor (IGF); Metalloprotease; Protease Inhibitor; Protein Complex; Transgenic Mice.

FIGURE 1.

Analysis of cultured MEFs derived from STC2 transgenic mice and nontransgenic littermates. A, proteolytic activity toward radiolabeled IGFBP-4 in medium conditioned by MEFs derived from STC2 transgenic mice or nontransgenic littermates (E13.5). Note that MEFs from Tg(STC2) mice contain only residual proteolytic activity. B, proteolytic activity toward radiolabeled IGFBP-4 in MEF medium derived from nontransgenic mice in the presence or absence of mAb 1/41 (32), a specific inhibitor of PAPP-A. C, co-immunoprecipitation (IP) of transgenic STC2 and endogenous PAPP-A from MEF medium derived from STC2 transgenic mice. Separation was done by nonreducing SDS-PAGE. WB, Western blot.

FIGURE 2.

PAPP-A activity is eliminated by cotransfection with STC2 cDNA but not by the addition of purified STC2 protein. A, PAPP-A proteolytic activity toward radiolabeled IGFBP-4 in media from HEK293T cells transfected with combinations of cDNAs as indicated. Cotransfection with STC2 cDNA appears to inhibit PAPP-A activity. B, PAPP-A Western blot (WB) of samples from A demonstrating similar levels of PAPP-A secretion from the transfected cells. Separation was done by reducing SDS-PAGE. C, PAPP-A proteolytic activity toward radiolabeled IGFBP-4 in the presence or absence of purified STC2 added at the beginning of the cleavage reaction. Note that in this experiment, STC2 did not inhibit PAPP-A.

FIGURE 3.

Inhibition of PAPP-A proteolytic activity by STC2 requires covalent complex formation. A, PAPP-A Western blot (WB) of medium from HEK293T cells transfected with combinations of cDNAs as indicated. Note the change in migration of PAPP-A upon cotransfection with STC2 cDNA. B, STC2 Western blot of samples from A. Note the appearance of a high molecular weight band upon cotransfection with PAPP-A cDNA. C, PAPP-A Western blot of separately synthesized PAPP-A and STC2 incubated for 0–16 h. SDS-PAGE separations for A–C were done under nonreducing conditions. D, PAPP-A proteolytic activity toward radiolabeled IGFBP-4 in 16-h samples from C.

FIGURE 4.

Cys-120 of STC2 is required for covalent complex formation and proteolytic inhibition. A, sequence alignment of human STC1 (NM_003155.2) and STC2 (NM_003714.2) using Clustal Omega. Disulfide bonds and dimerization disulfide of STC1, indicated by lines, are according to published data (38). Three cysteine residues of STC2 (Cys-120, Cys-197, and Cys-205), which have no counterpart in STC1, are indicated in blue. Cys-120 is conserved in all known mammalian STC2 sequences, and it has no counterpart in any known STC1 sequence (data not shown). B, STC2 Western blot following reducing SDS-PAGE of medium from HEK293T cells transfected with wild-type or mutated variants of STC2 cDNA as indicated. C, PAPP-A Western blot (WB) following nonreducing SDS-PAGE of media from HEK293T cells transfected with combinations of cDNAs as indicated. Note that STC2(C120A) is unable to form a covalent complex with PAPP-A. D, proteolytic activity toward radiolabeled IGFBP-4 in medium from HEK293T cells transfected with cDNA encoding PAPP-A, preincubated for 16 h with or without STC2 or STC2(C120A).

FIGURE 5.

STC2, but not STC2(C120A), abrogates PAPP-A-mediated IGF1R signaling in vitro. Cells expressing IGF1R were stimulated with combinations of IGF-I, IGFBP-4, PAPP-A, and STC2/STC2(C120A) as indicated, and cell lysates were analyzed by Western blotting (WB). Quantified signals of IGF receptor phosphorylation normalized to the signal with IGF-I alone are shown to the right. Results are means ± S.D. from four independent experiments (ns, not statistically significant; *, p < 0.0001).

FIGURE 6.

Biochemical properties of STC2 are retained in STC2(C120A). A, CD analysis of purified STC2 and STC2(C120A). Inset shows Coomassie-stained SDS-polyacrylamide gel of purified STC2 and STC2(C120A). B, CD spectra of STC2 and STC2(C120A) recorded at 25 and 90 °C and again at 25 °C following incubation at 90 °C for 2 min. C, relative binding of available monoclonal antibodies to STC2 and STC2(C120A) is plotted as a ratio between the levels measured in ELISAs. A value of 1 indicates equal binding to STC2 and STC2(C120A). Results are means ± S.D. from four independent experiments performed in triplicate. Differences in ratios are not statistically significant. D, proteolytic activity toward radiolabeled IGFBP-4 in medium from HEK293T cells transfected with cDNA encoding murine PAPP-A (mPAPP-A), preincubated with or without STC2 or STC2(C120A). E, Western blot (WB) following nonreducing SDS-PAGE of samples from D demonstrating covalent complex formation between murine PAPP-A and STC2 but not STC2(C120A).

FIGURE 7.

Overexpression of STC2, but not STC2(C120A), causes growth retardation in mice. A, growth curves of nontransgenic and STC2 transgenic female mice divided into groups according to circulating levels of STC2. Results are means with S.D. indicated. Statistical significance is based on comparison of mice older than 3 weeks (*, p < 0.02; **, p < 0.0001). The inset shows a transgenic mouse representative of the group with high (2–8 μg/ml) expression of STC2 (left) and a nontransgenic littermate. B, body weight of STC2 transgenic female mice at week 6 against measured levels of circulating transgene-derived STC2. C, growth curves of nontransgenic and STC2(C120A) transgenic female mice. The inset shows a transgenic mouse with high (2–8 μg/ml) expression of STC2(C120A) (left) and a nontransgenic littermate. Results are means with S.D. indicated. ns, not statistically significant.

FIGURE 8.

STC2 and PAPP-A, consequences of genetic manipulation in mice and working model of their role in the IGF system. A, size comparison of mice with targeted deletion of STC2 (28) or PAPP-A (17), or transgenic overexpression of STC2 (27) or STC2(C120A). B, model depicting the balance between active and STC2-inhibited PAPP-A, indirectly affecting IGF receptor (IGF1R) stimulation.