Production of recombinant porcine IGF-binding protein-5 and its effect on proliferation of porcine embryonic myoblast cultures in the presence and absence of IGF-I and Long-R3-IGF-I.
IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.
Pampusch
M S
MS
Animal Growth and Development Laboratory, Department of Animal Science, University of Minnesota, St Paul, Minnesota 55108, USA.
Xi
G
G
Kamanga-Sollo
E
E
Loseth
K J
KJ
Hathaway
M R
MR
Dayton
W R
WR
White
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000494:pharmacology; D016367:Baculoviridae; D001483:Base Sequence; D019149:Bioreactors; D015153:Blotting, Western / Q000379:methods; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D018076:DNA, Complementary / Q000032:analysis*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000196:embryology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013552:Swine
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D018975:Insulin-Like Growth Factor Binding Protein 5; D011994:Recombinant Proteins; C085700:long R(3)-insulin-like growth factor-I; D007334:Insulin-Like Growth Factor I