Elizabeth M. Wilson

Synthesis and Characterization of Insulin-like Growth Factor-binding Protein (IGFBP)-7 RECOMBINANT HUMAN mac25 PROTEIN SPECIFICALLY BINDS IGF-I AND -II

The mac25 cDNA was originally cloned from leptomeningial cells and subsequently reisolated through differential display as a sequence preferentially expressed in senescent human mammary epithelial cells. The deduced amino acid sequence of the human mac25 propeptide shares a 20-25% identity to human insulin-like growth factor-binding proteins (IGFBPs), suggesting that mac25 could be another member of the IGFBP family. In the present study, we have generated recombinant human mac25 (rh-mac25) in a baculovirus expression system and assessed its affinity for IGFs and have evaluated the pattern of expression of the mac25 gene in human tissues. Binding of 125I-IGF-I and 125I-IGF-II to rh-mac25 was demonstrated by Western ligand blotting after nondenaturing polyacrylamide gel electrophoresis and by affinity cross-linking with as little as 2 nM rh-mac25. Specificity of rh-mac25 binding to 125I-IGFs was demonstrated by competition for rh-mac25 binding with unlabeled IGFs, but not with [QAYLL]IGF-II analog, which has 100-fold less affinity for IGFBPs. In comparison with IGFBP-3, rh-mac25 has at least a 5-6-fold lower affinity for IGF-I and 20-25-fold lower affinity for IGF-II. mac25 mRNA was detectable in a wide range of normal human tissues, with decreased expression in breast, prostate, colon, and lung cancer cell lines. In conclusion, mac25 specifically binds IGFs and constitutes a new member of the IGFBP family, IGFBP-7. Its wider distribution in normal tissue and lower expression in several cancer cells indicate that IGFBP-7 may function as a growth-suppressing factor, as well as an IGF-binding protein.

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins.

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor β subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3(1–87)), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.

Insulin-like Growth Factor-mediated Muscle Differentiation COLLABORATION BETWEEN PHOSPHATIDYLINOSITOL 3-KINASE-Akt-SIGNALING PATHWAYS AND MYOGENIN

The differentiation and maturation of skeletal muscle require interactions between signaling pathways activated by hormones and growth factors and an intrinsic regulatory network controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo and in regeneration in the adult. To study mechanisms of IGF action in muscle, we developed a myogenic cell line that overexpresses IGF-binding protein-5. C2BP5 cells remain quiescent in low serum differentiation medium until the addition of IGF-I. Here we use this cell line to identify signaling pathways controlling IGF-mediated differentiation. Induction of myogenin by IGF-I and myotube formation were prevented by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, even when included 2 days after growth factor addition, whereas expression of active PI 3-kinase could promote differentiation in the absence of IGF-I. Differentiation also was induced by myogenin but was blocked by LY294002. The differentiation-promoting effects of IGF-I were mimicked by a modified membrane-targeted inducible Akt-1 (iAkt), and iAkt was able to stimulate differentiation of C2 myoblasts and primary mouse myoblasts incubated with otherwise inhibitory concentrations of LY294002. These results show that an IGF-regulated PI 3-kinase-Akt pathway controls muscle differentiation by mechanisms acting both upstream and downstream of myogenin.

β-catenin binds to the activation function 2 region of the androgen receptor and modulates the effects of the N-terminal domain and TIF2 on ligand-dependent transcription

ABSTRACT β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD). In functional assays, β-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists. β-Catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor. Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and β-catenin. β-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of β-catenin and not of TIF2 or the androgen receptor NTD. Mutational disruption of each of five LXXLL peptide motifs in the β-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation. ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to β-catenin also blocked binding of the androgen receptor LBD. We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of β-catenin and androgen receptor. Taken together, the data show that β-catenin can bind to the androgen receptor LBD and modulate the effects of the androgen receptor NTD and TIF2 on transcription.

Melanoma Antigen Gene Protein MAGE-11 Regulates Androgen Receptor Function by Modulating the Interdomain Interaction

ABSTRACT Gene activation by steroid hormone receptors involves the recruitment of the steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs to activation function 2 (AF2) in the ligand binding domain. For the androgen receptor (AR), AF2 also serves as the interaction site for the AR NH2-terminal FXXLF motif in the androgen-dependent NH2-terminal and carboxyl-terminal (N/C) interaction. The relative importance of the AR AF2 site has been unclear, since the AR FXXLF motif interferes with coactivator recruitment by competitive inhibition of LXXLL motif binding. In this report, we identified the X chromosome-linked melanoma antigen gene product MAGE-11 as an AR coregulator that specifically binds the AR NH2-terminal FXXLF motif. Binding of MAGE-11 to the AR FXXLF α-helical region stabilizes the ligand-free AR and, in the presence of an agonist, increases exposure of AF2 to the recruitment and activation by the SRC/p160 coactivators. Intracellular association between AR and MAGE-11 is supported by their coimmunoprecipitation and colocalization in the absence and presence of hormone and by competitive inhibition of the N/C interaction. AR transactivation increases in response to MAGE-11 and the SRC/p160 coactivators through mechanisms that include but are not limited to the AF2 site. MAGE-11 is expressed in androgen-dependent tissues and in prostate cancer cell lines. The results suggest MAGE-11 is a unique AR coregulator that increases AR activity by modulating the AR interdomain interaction.

Selective Control of Skeletal Muscle Differentiation by Akt1

The phosphatidylinositol 3-kinase-Akt pathway plays a central role in growth, development, and metabolism in both normal and neoplastic cells. In skeletal muscle, Akt has been implicated in regulating regeneration and hypertrophy and in counteracting atrophy. Here we provide evidence that Akt1 and not Akt2 is essential for muscle differentiation. Using a robust model of MyoD-mediated muscle development, in which dominant-negative Akt blocked differentiation, we show that targeted loss of Akt1 was equally inhibitory. Selective elimination of Akt1 had no effect on myoblast viability or proliferation but prevented differentiation by impairing the transcriptional actions of MyoD. In contrast, knockdown of Akt2 had no effect on myoblast survival or differentiation and minimally inhibited MyoD-regulated transcription. Our results define isoform-specific Akt-regulated signaling pathways in muscle cells that act through Akt1 to sustain muscle gene activation and promote differentiation.

Control of MyoD function during initiation of muscle differentiation by an autocrine signaling pathway activated by insulin- like growth factor-II

The insulin-like growth factors (IGFs) play key roles in muscle development, maintenance, and repair, but their mechanisms of action are incompletely defined. We previously identified an autocrine pathway involving production of IGF-II and activation of the IGF-I receptor, phosphatidylinositol 3-kinase, and Akt in myoblast differentiation induced by MyoD in 10T1/2 mesenchymal stem cells and found that blocking this pathway prevented differentiation (Wilson, E. M., Hsieh, M. M., and Rotwein, P. (2003) J. Biol. Chem. 278, 41109-41113). We now have analyzed regulation of MyoD function in this model system. Inhibition of IGF-II production impaired the transcriptional actions of MyoD, as seen by a 70-80% decline in activity of transfected reporter genes, including the myogenin and creatine kinase promoters, and by complete inhibition of transcription of the endogenous myogenin gene but had no effect on MyoD protein levels, post-translational modifications, or nuclear localization, and neither blocked the rapid disappearance of the inhibitory molecule Id1 nor altered the nuclear expression or abundance of the MyoD binding partner E12/E47. Impaired signaling through the IGF-I receptor also did not decrease the ability of MyoD or E12/E47 to bind to target DNA sites at the proximal myogenin promoter, as assessed by chromatin immunoprecipitation assay but, rather, blocked chromatin remodeling at this site, as indicated by reduced recruitment of co-activators p300 and P/CAF and diminished acetylation of histones H3 and H4. Taken together, these results show that IGF-II-initiated signaling through the insulin-like growth factor-I receptor targets transcriptional co-regulators that are essential co-factors for MyoD and suggests that the phosphatidylinositol 3-kinase-Akt pathway plays a key role in establishing an amplification cascade that is essential for sustaining the earliest events in muscle differentiation.

Electrostatic modulation in steroid receptor recruitment of LXXLL and FXXLF motifs.

ABSTRACT Coactivator recruitment by activation function 2 (AF2) in the steroid receptor ligand binding domain takes place through binding of an LXXLL amphipathic α-helical motif at the AF2 hydrophobic surface. The androgen receptor (AR) and certain AR coregulators are distinguished by an FXXLF motif that interacts selectively with the AR AF2 site. Here we show that LXXLL and FXXLF motif interactions with steroid receptors are modulated by oppositely charged residues flanking the motifs and charge clusters bordering AF2 in the ligand binding domain. An increased number of charged residues flanking AF2 in the ligand binding domain complement the two previously characterized charge clamp residues in coactivator recruitment. The data suggest a model whereby coactivator recruitment to the receptor AF2 surface is initiated by complementary charge interactions that reflect a reversal of the acidic activation domain-coactivator interaction model.

Selective signaling by Akt2 promotes bone morphogenetic protein 2-mediated osteoblast differentiation

ABSTRACT Mesenchymal stem cells are essential for repair of bone and other supporting tissues. Bone morphogenetic proteins (BMPs) promote commitment of these progenitors toward an osteoblast fate via functional interactions with osteogenic transcription factors, including Dlx3, Dlx5, and Runx2, and also can direct their differentiation into bone-forming cells. BMP-2-stimulated osteoblast differentiation additionally requires continual signaling from insulin-like growth factor (IGF)-activated pathways. Here we identify Akt2 as a critical mediator of IGF-regulated osteogenesis. Targeted knockdown of Akt2 in mouse primary bone marrow stromal cells or in a mesenchymal stem cell line, or genetic knockout of Akt2, did not interfere with BMP-2-mediated signaling but resulted in inhibition of osteoblast differentiation at an early step that preceded production of Runx2. In contrast, Akt1-deficient cells differentiated normally. Complete biochemical and morphological osteoblast differentiation was restored in cells lacking Akt2 by adenoviral delivery of Runx2 or by a recombinant lentivirus encoding wild-type Akt2. In contrast, lentiviral Akt1 was ineffective. Taken together, these observations define a specific role for Akt2 as a gatekeeper of osteogenic differentiation through regulation of Runx2 gene expression and indicate that the closely related Akt1 and Akt2 exert distinct effects on the differentiation of mesenchymal precursors.

Distinct actions of Akt1 and Akt2 in skeletal muscle differentiation

Differentiation, maturation, and repair of skeletal muscle requires ongoing cooperation between signaling cascades activated by hormones and growth factors, and intrinsic regulatory programs controlled by myogenic transcription factors. The insulin‐like growth factor—phosphatidylinositol‐3 kinase—Akt pathway has been implicated in muscle growth and regeneration after injury, in counteracting sarcopenia during aging, and in maintaining muscle cell viability. Here we present evidence for distinct roles for Akt1 and Akt2 in different phases of muscle differentiation. Targeted knockdown of either Akt had no effect on C2 myoblast proliferation, even though Akt1 concentrations are markedly higher than Akt2 levels under growth‐promoting conditions. Akt2 concentrations rose by nearly an order of magnitude during muscle differentiation, while Akt1 levels remained constant, yet loss of either protein did not increase myoblast death. Rather, knockdown or genetic knockout of Akt1 blocked differentiation at its earliest stages, preventing induction of muscle‐specific proteins and inhibiting formation of multinucleated myofibers, while myoblasts lacking Akt2 differentiated normally, although resultant myofibers were thinner and incorporated fewer nuclei than controls. Forced expression of knockdown‐resistant Akt1 partially reversed the deficit in differentiation seen in myoblasts lacking Akt1. Our results define isoform‐specific Akt actions in muscle cells, and demonstrate that both Akts are necessary for full myoblast differentiation and maturation. J. Cell. Physiol. 219: 503–511, 2009.