William R. Dayton

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.

A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low-calcium-requiring form of the protease.

Two forms of calcium-activated neutral protease were isolated and purified from porcine skeletal muscle. The two forms of the protease differ markedly in their requirement for calcium with the low-calcium-requiring form showing one-half maximal activation at 45 micro M calcium while the high-calcium-requiring form shows one-half maximal activation at 0.74 micro M calcium. Additionally, they chromatograph differently on DEAE-cellulose, exhibit different mobilities in electrophoresis in a nondenaturing buffer, are affected differently by certain divalent cations, and have slightly different pH dependencies. Despite these differences, the purified forms of the calcium-activated protease co-chromatograph in gel permeation chromatography, have identical banding patterns on sodium dodecyl sulfate (SDS)-polyacrylamide gels, cross-react with an antibody directed against the 80 000-dalton subunit of the calcium-activated protease we originally purified from skeletal muscle (Dayton, W.R., Goll, D.E., Zeece, M.G., Robson, R.M. and Reville, W.J. (1976) Biochemistry 15, 2150-2158), and have identical effects on the ultrastructure of myofibrils. THe high-calcium-requiring protease purified in this study is very likely identical to the calcium-activated protease we originally purified from skeletal muscle. The properties of the low-calcium-requiring form of the protease suggest that it is the form of the enzyme that is active in vivo.

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.

Comparison of low- and high-calcium-requiring forms of the calcium-activated protease with their autocatalytic breakdown products

The effect of controlled autocatalytic degradation on the subunit molecular weight, subunit composition and calcium sensitivity of the low-calcium-requiring and the high-calcium-requiring forms of the calcium-activated muscle protease was examined. Purified low- and high-calcium-requiring proteases coelectrophorese on SDS-polyacrylamide gels. Controlled autocatalysis of either form of the protease results in extensive degradation of their respective 30 kDa subunits and partial degradation of their 80 kDa subunits. In this electrophoresis system the electrophoretic banding pattern of the low-calcium-requiring protease is clearly different from that of the autocatalytically degraded high-calcium-requiring protease. Similar results were obtained using a nondenaturing polyacrylamide gel system. Both the high- and low-calcium-requiring proteases were made more sensitive to calcium by autocatalytic degradation. However, the results of this study strongly indicate that autocatalytic degradation does not result in conversion of high-calcium-requiring protease to the low-calcium-requiring protease as has been recently hypothesized (Suzuki et al. (1981) J. Biochem. 90, 275-278).

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

Abstract The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.

Elevated levels of a calcium-activated muscle protease in rapidly atrophying muscles from vitamin E-deficient rabbits

A Ca2+-activated proteolytic enzyme that partially degrades myofibrils was isolated from hind limb muscles of normal rabbits and rabbits undergoing rapid muscle atrophy as a result of vitamin E deficiency. Extractable Ca2+-activated protease activity was 3.6 times higher in muscle tissue from vitamin E-deficient rabbits than from muscle tissue of control rabbits. Ultrastructural studies of muscle from vitamin E-deficient rabbits showed that the Z disk was the first myofibrillar structure to show degradative changes in atrophying muscle. Myofibrils prepared from muscles from vitamin E-deficient rabbits showed partial or complete loss of Z-disk density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the amount of troponin-T (37 000 daltons) and alpha-actinin (96 000 daltons) was reduced in myofibrils from atrophying muscle as compared to myofibrils prepared from control muscle. In vitro treatment of purified myofibrils with purified Ca2+-activated proteolytic enzyme produced alterations in myofibrillar ultrastructure that were identical to the initial alterations occurring in myofibrils from atrophying muscle (i.e. weakening and subsequent removal of Z disks). Additonally the electrophoretic banding pattern of Ca2+-activated proteolytic enzyme-treated myofibrils is very similar to that of myofibrils prepared from muscles atrophying as a result of nutritional vitamin E deficiency. The possible role of Ca2+-activated proteolytic enzyme in disassembly and degradation of the myofibril is discussed.

Isolation from porcine cardiac muscle of a Ca2+-activated protease that partially degrades myofibrils.

Abstract A protein fraction displaying Ca 2+ -activated proteolytic activity has been isolated from porcine cardiac muscle. The crude enzyme was purified approximately 2000 fold by isoelectric precipitation followed by gel permeation chromatography and by ion exchange chromatography. The partially purified enzyme exhibited optimal activity against either cardiac myofibril or casein substrates between pH 7.5 and 8.0, and in the presence of 1 m m Ca 2+ and at least 2 m m 2-mercaptoethanol. The enzyme removes Z-discs from skeletal and cardiac myofibrils and also removes the density from intercalated discs of cardiac myofibrils. The enzyme hydrolyzes troponin-T and troponin-I of both cardiac and skeletal muscle myofibrils in vitro . In its proteolytic effect on either cardiac or skeletal myofibrils and in all other properties examined, the Ca 2+ -activated, cardiac protease is similar to a Ca 2+ -activated protease (CAF) recently purified from porcine skeletal muscle (Dayton, W. R., Reville, W. J., Goll, D. E. and Stromer, M. H. (1976) Biochemistry 15 , 2159–2167). It is possible that the Ca 2+ -activated, cardiac protease plays a role in degradation of myofibrils in injured myocardial cells.

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.