Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3)

Tomonobu Hasegawa, P. Cohen, Y. Hasegawa, P. J. Fielder, R. G. Rosenfeld

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Characterization of the insulin-like growth factor (IGF) axis in a cultured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayable IGF-I and IGF-II concentrations in TM-3 conditioned media (CM) were below the assay detection levels. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from TM-3 cells revealed both type 1 and type 2 receptors and a 31 kDa IGF binding protein (IGFBP). Immunoprecipitation of solubilized crude membranes indicated that the 31 kDa protein was membrane associated IGFBP-4. Western ligand blots of CM from TM-3 cells demonstrated the presence of 24 and 28 kDa bands as major IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band could be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa bands were immunoprecipitated with an antibody to IGFBP-4. Treatment of TM-3 cells with IGF-I increased the levels of the 24 kDa IGFBP-4, as well as 25, 28, 40, and 44 kDa IGFBPs. IGF-I treatment also resulted in the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands were deglycosylated with Endoglycosidase-F nor immunoprecipitated by antibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment resulted in a significant decrease in the 24 kDa IGFBP-4. When [Leu27]IGF-II, which binds to the type 2 receptor and to IGFBPs, but not to the type 1 receptor, was added to the cells, it mimicked the effect of IGF-II on the 24 kDa IGFBP-4. Although both IGF-I and -II affected the level of 24 kDa IGFBP-4 in CM, neither peptide affected the expression of messenger RNA for IGFBP-4. Northern blot analysis of total RNA prepared from TM-3 cells treated with IGF-I shared the expression of mRNA for IGFBP-5, which was not seen in untreated cells, suggesting that the 29 kDa IGFBP seen in the CM of TM-3 cells treated with IGF-I, might be IGFBP-5. The presence of mRNA for IGFBP-6 was noted under basal conditions. In conclusion, neither IGF-I nor IGF-II was detectable in TM-3 CM. TM-3 cells have type 1 and type 2 IGF receptors and membrane associated IGFBP-4. TM-3 cells can produce IGFBP-4 and glycosylated IGFBP-4 as major IGFBPs, and 40 and 44 kDa IGFBPs and a 25 kDa IGFBP (presumably IGFBP-6) under basal conditions. Treatment with IGF-I increased levels of a 29 kDa IGFBP (presumably IGFBP-5). The divergent effects of IGF-I and -II on the concentrations of IGFBP-4 in CM are post-transcriptional.

Original languageEnglish
Pages (from-to)151-159
Number of pages9
JournalGrowth Regulation
Volume5
Issue number3
Publication statusPublished - 1995
Externally publishedYes

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Insulin-Like Growth Factor Binding Protein 4
Insulin-Like Growth Factor Binding Proteins
Leydig Cells
Somatomedins
Insulin-Like Growth Factor I
Insulin-Like Growth Factor II
Cell Line
Conditioned Culture Medium
Insulin-Like Growth Factor Binding Protein 5
Insulin-Like Growth Factor Binding Protein 6
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Messenger RNA
Membrane Proteins
IGF Type 2 Receptor
IGF Type 1 Receptor
RNA-Binding Proteins
Membranes
Antibodies
Immunoprecipitation
Northern Blotting

Keywords

  • IGF-I
  • IGF-II
  • IGFBP-4
  • Membrane associated IGFBP-4
  • Post-transcriptional
  • TM-3 cells
  • Type 1 receptor
  • Type 2 receptor

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3). / Hasegawa, Tomonobu; Cohen, P.; Hasegawa, Y.; Fielder, P. J.; Rosenfeld, R. G.

In: Growth Regulation, Vol. 5, No. 3, 1995, p. 151-159.

Research output: Contribution to journalArticle

Hasegawa, T, Cohen, P, Hasegawa, Y, Fielder, PJ & Rosenfeld, RG 1995, 'Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3)', Growth Regulation, vol. 5, no. 3, pp. 151-159.
Hasegawa, Tomonobu ; Cohen, P. ; Hasegawa, Y. ; Fielder, P. J. ; Rosenfeld, R. G. / Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3). In: Growth Regulation. 1995 ; Vol. 5, No. 3. pp. 151-159.
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T1 - Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3)

AU - Hasegawa, Tomonobu

AU - Cohen, P.

AU - Hasegawa, Y.

AU - Fielder, P. J.

AU - Rosenfeld, R. G.

PY - 1995

Y1 - 1995

N2 - Characterization of the insulin-like growth factor (IGF) axis in a cultured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayable IGF-I and IGF-II concentrations in TM-3 conditioned media (CM) were below the assay detection levels. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from TM-3 cells revealed both type 1 and type 2 receptors and a 31 kDa IGF binding protein (IGFBP). Immunoprecipitation of solubilized crude membranes indicated that the 31 kDa protein was membrane associated IGFBP-4. Western ligand blots of CM from TM-3 cells demonstrated the presence of 24 and 28 kDa bands as major IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band could be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa bands were immunoprecipitated with an antibody to IGFBP-4. Treatment of TM-3 cells with IGF-I increased the levels of the 24 kDa IGFBP-4, as well as 25, 28, 40, and 44 kDa IGFBPs. IGF-I treatment also resulted in the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands were deglycosylated with Endoglycosidase-F nor immunoprecipitated by antibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment resulted in a significant decrease in the 24 kDa IGFBP-4. When [Leu27]IGF-II, which binds to the type 2 receptor and to IGFBPs, but not to the type 1 receptor, was added to the cells, it mimicked the effect of IGF-II on the 24 kDa IGFBP-4. Although both IGF-I and -II affected the level of 24 kDa IGFBP-4 in CM, neither peptide affected the expression of messenger RNA for IGFBP-4. Northern blot analysis of total RNA prepared from TM-3 cells treated with IGF-I shared the expression of mRNA for IGFBP-5, which was not seen in untreated cells, suggesting that the 29 kDa IGFBP seen in the CM of TM-3 cells treated with IGF-I, might be IGFBP-5. The presence of mRNA for IGFBP-6 was noted under basal conditions. In conclusion, neither IGF-I nor IGF-II was detectable in TM-3 CM. TM-3 cells have type 1 and type 2 IGF receptors and membrane associated IGFBP-4. TM-3 cells can produce IGFBP-4 and glycosylated IGFBP-4 as major IGFBPs, and 40 and 44 kDa IGFBPs and a 25 kDa IGFBP (presumably IGFBP-6) under basal conditions. Treatment with IGF-I increased levels of a 29 kDa IGFBP (presumably IGFBP-5). The divergent effects of IGF-I and -II on the concentrations of IGFBP-4 in CM are post-transcriptional.

AB - Characterization of the insulin-like growth factor (IGF) axis in a cultured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayable IGF-I and IGF-II concentrations in TM-3 conditioned media (CM) were below the assay detection levels. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from TM-3 cells revealed both type 1 and type 2 receptors and a 31 kDa IGF binding protein (IGFBP). Immunoprecipitation of solubilized crude membranes indicated that the 31 kDa protein was membrane associated IGFBP-4. Western ligand blots of CM from TM-3 cells demonstrated the presence of 24 and 28 kDa bands as major IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band could be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa bands were immunoprecipitated with an antibody to IGFBP-4. Treatment of TM-3 cells with IGF-I increased the levels of the 24 kDa IGFBP-4, as well as 25, 28, 40, and 44 kDa IGFBPs. IGF-I treatment also resulted in the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands were deglycosylated with Endoglycosidase-F nor immunoprecipitated by antibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment resulted in a significant decrease in the 24 kDa IGFBP-4. When [Leu27]IGF-II, which binds to the type 2 receptor and to IGFBPs, but not to the type 1 receptor, was added to the cells, it mimicked the effect of IGF-II on the 24 kDa IGFBP-4. Although both IGF-I and -II affected the level of 24 kDa IGFBP-4 in CM, neither peptide affected the expression of messenger RNA for IGFBP-4. Northern blot analysis of total RNA prepared from TM-3 cells treated with IGF-I shared the expression of mRNA for IGFBP-5, which was not seen in untreated cells, suggesting that the 29 kDa IGFBP seen in the CM of TM-3 cells treated with IGF-I, might be IGFBP-5. The presence of mRNA for IGFBP-6 was noted under basal conditions. In conclusion, neither IGF-I nor IGF-II was detectable in TM-3 CM. TM-3 cells have type 1 and type 2 IGF receptors and membrane associated IGFBP-4. TM-3 cells can produce IGFBP-4 and glycosylated IGFBP-4 as major IGFBPs, and 40 and 44 kDa IGFBPs and a 25 kDa IGFBP (presumably IGFBP-6) under basal conditions. Treatment with IGF-I increased levels of a 29 kDa IGFBP (presumably IGFBP-5). The divergent effects of IGF-I and -II on the concentrations of IGFBP-4 in CM are post-transcriptional.

KW - IGF-I

KW - IGF-II

KW - IGFBP-4

KW - Membrane associated IGFBP-4

KW - Post-transcriptional

KW - TM-3 cells

KW - Type 1 receptor

KW - Type 2 receptor

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