E. Kamanga-Sollo

Roles of IGF-I and the estrogen, androgen and IGF-I receptors in estradiol-17β- and trenbolone acetate-stimulated proliferation of cultured bovine satellite cells

Although numerous studies have shown that both androgenic and estrogenic steroids increase rate and efficiency of muscle growth in steers, there is little consensus as to their mechanism of action. A combined estradiol 17beta (E2)/trenbolone acetate (TBA) implant causes a significant increase in muscle IGF-I mRNA and both E2 and TBA stimulate a significant increase in IGF-I mRNA level in bovine satellite cell (BSC) cultures in media containing 10% fetal bovine serum (FBS). Consequently, increased IGF-I expression may play a role in anabolic-steroid-enhanced muscle growth. However, even though treatment of cultured BSC with E2 or TBA in media containing 1% IGFBP-3-free swine serum (SS) results in increased proliferation there is no effect on IGF-I mRNA expression, suggesting that increased IGF-I expression may not be responsible for anabolic-steroid-enhanced BSC proliferation. To further examine the role of estrogen, androgen and IGF-I receptors and their respective ligands in E2- and TBA-stimulated BSC proliferation, we assessed the effects of specific inhibitors on E2- or TBA-stimulated proliferation of BSC. Both ICI 182 780 (an estrogen receptor blocker) and flutamide (an inhibitor of androgen receptor) suppressed (p<0.05) E2- and TBA-stimulated BSC proliferation, respectively. JB1 (a competitive inhibitor of IGF-I binding to type I IGF receptor) reduced (p<0.05) both E2- and TBA-stimulated proliferation in BSC cultures. Both the Raf-1/MAPK kinase (MEK)1/2/ERK1/2, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways play significant roles in the actions of IGF-I on proliferation and differentiation of myogenic cells. PD98059, an inhibitor of the MAPK pathway, and wortmannin, an inhibitor of the PI3K pathway, both suppressed (p<0.05) E2- and TBA-stimulated proliferation of cultured BSC. Our data suggest that IGF-I plays a role in E2- and TBA-stimulated proliferation of cultured BSC even in the absence of increased IGF-I expression.

IGF-I mRNA levels in bovine satellite cell cultures: Effects of fusion and anabolic steroid treatment

Androgenic and estrogenic steroids enhance muscle growth in a number of species; however, the mechanism by which anabolic steroids enhance muscle growth is not known. Castrated male cattle (steers) provide a particularly good model system in which to study the effects of anabolic steroids on muscle growth because they respond dramatically to treatment with both estrogens and androgens. The goal of this study was to determine if treatment of bovine satellite cell (BSC) cultures with 17β‐estradiol (E2) or trenbolone (a synthetic androgen) directly affects proliferation rate or level of mRNA for estrogen receptor (ER)‐α, androgen receptor, and growth factors that have been shown to affect muscle growth (insulin‐like growth factor (IGF)‐I, IGF binding protein (IGFBP)‐3, and myostatin). BSC cultures were established from the semimembranosus muscles of steers and then treated for 48 h with various concentrations of E2 or trenbolone ranging from 0.001 to 10 nM. IGF‐I mRNA levels in proliferating BSC cultures were significantly increased at 0.01 (1.9‐times control values, P < 0.02) and at 0.1, 1, and 10 nM E2 (2.9‐, 3.5‐, and 3.5‐times control values, respectively, P < 0.0001). Additionally both 1 and 10 nM trenbolone increased IGF‐I mRNA levels to 1.7‐times control values (P < 0.02). ER‐α mRNA was detectable in BSC cultures, and levels were increased (2.3‐times control levels, P < 0.001) in cultures treated with 0.001 nM E2 but not in cultures treated with higher concentrations of E2. Androgen receptor mRNA levels also were increased (1.5‐times control levels, P < 0.02) in cultures treated with 0.001 nM trenbolone but not by treatment with higher concentrations of trenbolone. Levels of IGFBP‐3 were increased (1.4‐times control values, P < 0.02) by treatment with 0.001 nM E2 but not by treatment with high concentrations of E2. Myostatin mRNA levels were not affected by any concentration of either of the steroids. Although, levels of IGF‐I mRNA were 10‐times greater (P < 0.02) in fused BSC cultures than in proliferating cultures, treatment of fused cultures for 48 h with 10 nM E2 increased IGF‐I mRNA levels (2.5‐times control levels, P < 0.02). Both E2 and trenbolone increased 3H‐thymidine incorporation rate (1.5‐times control levels, P < 0.001) in BSC cultures in media containing serum from which IGFBP‐3 had been removed by anti‐IGFBP‐3 affinity chromatography. In summary, treatment of BSC cultures with either E2 or trenbolone increased IGF‐I mRNA level and proliferation rate, thus, establishing that these steroids have direct anabolic effects on cells present in the BSC culture.

Potential role of G-protein-coupled receptor 30 (GPR30) in estradiol-17β-stimulated IGF-I mRNA expression in bovine satellite cell cultures

Androgenic and estrogenic steroids enhance muscle growth in animals and humans. Estradiol-17beta (E2) and trenbolone acetate (TBA) (a synthetic testosterone analog) increased IGF-I mRNA expression in bovine muscle satellite cell (BSC) cultures. The goal of this study was to evaluate the mechanisms responsible for this increase by evaluating the effects of ICI 182 780 (an E2 receptor antagonist), flutamide (an androgen receptor inhibitor), G1 (a GPR30 agonist), and BSA-conjugated E2 on E2 and/or TBA-stimulated IGF-I mRNA expression in BSC cultures. Flutamide completely suppressed TBA-stimulated IGF-I mRNA expression in BSC cultures. ICI 182 780 did not suppress E2-stimulated IGF-I mRNA expression and 100 nM ICI 182 780 enhanced (93%, p<0.05) IGF-I mRNA levels in BSC cultures. G1 (100 nM) stimulated IGF-I mRNA expression (100%, p<0.05) but had no effect on proliferation in BSC cultures. E2-BSA, which cannot cross the cell membrane, stimulated IGF-I mRNA expression (approximately 100%, p<0.05) in BSC but even at extremely high concentrations had no effect on proliferation. In summary, our data indicate the E2-stimulation of proliferation and E2-stimulation of IGF-I mRNA expression in BSC cultures occur via different mechanisms. Our previous results showing that ICI 182 780 inhibited BSC proliferation and results of the current study showing lack of response to E2-BSA or G1 suggest that E2-stimulated proliferation in BSC cultures is mediated through classical estrogen receptors. Stimulation by ICI 182 780, G1 and E2-BSA suggests the E2-stimulated IGF-I mRNA expression in BSC cultures is mediated through the GPR30 receptor.

Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-β- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures

Both transforming growth factor (TGF‐β) and growth and development factor (GDF)‐8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells. In contrast, insulin‐like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells. In vivo, IGFs are found in association with a family of high‐affinity insulin‐like growth factor binding proteins (IGFBP 1–6) that affect their biological activity. Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF‐β1 or GDF‐8 suppressed proliferation and increased production of IGFBP‐3 protein and mRNA (P < 0.005). An anti‐IGFBP‐3 antibody that neutralizes the biological activity of IGFBP‐3 reduced the ability of either TGF‐β1 or GDF‐8 to suppress PEMC proliferation (P < 0.005). However, this antibody did not affect proliferation rate in the presence of both TGF‐β1 and GDF‐8. These data show that IGFBP‐3 plays a role in mediating the activity of either TGF‐β1 or GDF‐8 alone but not when both TGF‐β1 and GDF‐8 are present. In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP‐3 did not result in increased levels of phosphosmad‐2. Since TGF‐β and GDF‐8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP‐3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP‐3 also may be involved in regulating these processes in myogenic cells. J. Cell. Physiol. 197: 225–231, 2003© 2003 Wiley‐Liss, Inc.

Effect of Estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cultures

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Further, in vivo studies indicate that estradiol (E2) affects muscle protein synthesis and/or degradation, but in vitro results are inconsistent. We have examined the effects of E2 treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, to learn more about the mechanisms involved in E2-enhanced muscle growth, we have examined the effects of compounds that interfere with binding of E2 or insulin-like growth factor (IGF)-1 to their respective receptors on E2-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in protein degradation rate. The pure estrogen antagonist ICI 182 780 suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation in fused BSC cultures. The G-protein coupled receptor (GPR)-30 agonist G1 does not affect either synthesis or degradation rate, which establishes that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-1 binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation. In summary, our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical estrogen receptor (ER) and IGFR-1.

Effect of recombinant porcine IGFBP-3 on IGF-I and long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells

Insulin‐like growth factor (IGF)‐I stimulates both proliferation and differentiation of myogenic precursor cells. In vivo, IGFs are bound to one of the members of a family of six high‐affinity IGF binding proteins (IGFBP 1–6) that regulate their biological activity. One of these binding proteins, IGFBP‐3, affects cell proliferation via both IGF‐dependent and IGF‐independent mechanisms and it has generally been shown to suppress proliferation of cultured cells; however, it also may stimulate proliferation depending upon the cell type and the assay conditions. Cultured porcine embryonic myogenic cells (PEMCs) produce IGFBP‐3 and its level drops significantly immediately prior to differentiation. Additionally, IGFBP‐3 suppresses both IGF‐I and Long‐R3‐IGF‐I‐stimulated proliferation of embryonic porcine myogenic cells. In this study, we have examined the effects of recombinant porcine IGFBP‐3 (rpIGFBP‐3) on IGF‐I‐ and Long‐R3‐IGF‐I‐stimulated proliferation and differentiation of the L6 myogenic cell line. L6 cells potentially provide a good model for studying the actions of IGFBP‐3 on muscle because they contain no non‐muscle cells and they do not produce detectable levels of IGFBP‐3. RpIGFBP‐3 suppresses both IGF‐I and Long‐R3‐IGF‐I‐stimualted proliferation of L6 cells, indicating that it suppresses proliferation via both IGF‐dependent and IGF‐independent mechanisms. Our data also show that rpIGFBP‐3 causes IGF‐independent suppression of proliferation without increasing the level of phosphosmad‐2 in L6 cultures. Additionally, rpIGFBP‐3 suppresses IGF‐I‐stimulated differentiation of L6 cells. In contrast, however, rpIGFBP‐3 does not suppress Long‐R3‐IGF‐I‐stimulated differentiation. This suggests that rpIGFBP‐3 does not have IGF‐independent effects on L6 cell differentiation.

Effects of heat stress on proliferation, protein turnover, and abundance of heat shock protein messenger ribonucleic acid in cultured porcine muscle satellite cells

It is well established that heat stress (HS) negatively affects growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other nonswine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. Evidence is now emerging that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating the rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on quantities of Hsp90, Hsp70, and Hsp25/27 mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P < 0.05) in the abundance of Hsp90, Hsp70, and Hsp25/27 mRNA, respectively, relative to control cultures. Abundance of Hsp 90, 70, and 25/27 proteins was also increased in HS PSC cultures compared with those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P < 0.05) than those in control cultures. Protein synthesis rates in HS-fused PSC cultures were 85% greater (P < 0.05) than those in control cultures, and protein degradation rates in HS-fused PSC were 23% less (P < 0.05) than those in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and abundance of Hsp mRNA and Hsp protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine.

Role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in estradiol-stimulated proliferation of cultured bovine satellite cells

Although the exact mechanism(s) by which estradiol (E(2)) enhances muscle growth in a number of species, including humans and cattle, is not known, E(2) treatment has been shown to stimulate proliferation of cultured bovine satellite cells (BSCs). This is particularly significant because satellite cells are the source of nuclei needed to support postnatal muscle fiber hypertrophy and are thus crucial in determining the rate and extent of muscle growth. The objective of this study was to assess the role of estrogen receptor-α (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in E(2)-stimulated proliferation of cultured BSCs. To accomplish this, we have used small interfering RNA (siRNA) to silence expression of ESR1 or IGFR1 and assessed the effects on E(2)-stimulated proliferation in BSC cultures. In BSCs treated with nonspecific siRNA, E(2) significantly (P < 0.05) stimulates proliferation under conditions in which neither IGF-1 nor IGF-2 expression is increased; however, treatment of ESR1- or IGFR1-silenced cells with E(2) does not significantly stimulate proliferation. These results indicate that both ESR1 and IGFR1 are required for E(2) to stimulate proliferation in BSC cultures. The fact that this occurs under culture conditions in which neither IGF-1 nor IGF-2 mRNA expression is increased strongly suggests that E(2) activates IGFR1 via a mechanism that does not involve increased IGF-1 or IGF-2 binding to the receptor.

Effect of trenbolone acetate on protein synthesis and degradation rates in fused bovine satellite cell cultures

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Although in vivo studies have indicated that androgens affect protein synthesis and protein degradation rate in muscle, results from in vitro studies have been inconsistent. We have examined the effects of trenbolone acetate (TBA), a synthetic androgen, on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, we have examined the effects of compounds that interfere with binding of TBA or insulin-like growth factor-1 (IGF-1) to their respective receptors on TBA-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with TBA results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in degradation rate, establishing that TBA directly affects these parameters. Flutamide, a compound that prevents androgen binding to the androgen receptor, suppresses (P < 0.05) TBA-induced alterations in protein synthesis and degradation in fused BSC cultures, indicating the androgen receptor is involved. JB1, a competitive inhibitor of IGF-1 binding to the type 1 IGF receptor (IGF1R), suppresses (P < 0.05) TBA-induced alterations in protein synthesis and degradation, indicating that this receptor also is involved in the actions of TBA on both synthesis and degradation. In summary, our data show that TBA acts directly to alter both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the androgen receptor and IGF1R.

Role of G protein-coupled estrogen receptor-1, matrix metalloproteinases 2 and 9, and heparin binding epidermal growth factor-like growth factor in estradiol-17β-stimulated bovine satellite cell proliferation.

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters. Although the positive effects of E2 on rate and efficiency of bovine muscle growth are well established, the mechanisms involved in these effects are not well understood. Combined E2 and trenbolone acetate implants result in significantly increased muscle satellite cell number in feedlot steers. Additionally, E2 treatment stimulates proliferation of cultured bovine satellite cells (BSC). Studies in nonmuscle cells have shown that binding of E2 to G protein-coupled estrogen receptor (GPER)-1 results in activation of matrix metalloproteinases 2 and 9 (MMP2/9) resulting in proteolytic release of heparin binding epidermal growth factor-like growth factor (hbEGF) from the cell surface. Released hbEGF binds to and activates the epidermal growth factor receptor resulting in increased proliferation. To assess if GPER-1, MMP2/9, and/or hbEGF are involved in the mechanism of E2-stimulated BSC proliferation, we have examined the effects of G36 (a specific inhibitor of GPER-1), CRM197 (a specific inhibitor of hbEGF), and MMP-2/MMP-9 Inhibitor II (an inhibitor of MMP2/9 activity) on E2-stimulated BSC proliferation. Inhibition of GPER-1, MMP2/9, or hbEGF suppresses E2-stimulated BSC proliferation (P < 0.001) suggesting that all these are required in order for E2 to stimulate BSC proliferation. These results strongly suggest that E2 may stimulate BSC proliferation by binding to GPER-1 resulting in MMP2/9-catalyzed release of cell membrane-bound hbEGF and subsequent activation of epidermal growth factor receptor by binding of released hbEGF.