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Endocrinology, doi:10.1210/en.2005-1269
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Endocrinology Vol. 147, No. 5 2138-2146
Copyright © 2006 by The Endocrine Society

Ribonucleic Acid Polymerase II Binding Subunit 3 (Rpb3), a Potential Nuclear Target of Insulin-Like Growth Factor Binding Protein-3

Mohammed Oufattole, Sally Wan-Jung Lin, Bingrong Liu, Desmond Mascarenhas, Pinchas Cohen and Buel D. Rodgers

Department of Animal Sciences (M.O., S.W.-J.L., B.D.R.), Washington State University, Pullman, Washington 99164; Division of Pediatric Endocrinology (B.L., P.C.), Mattel Children’s Hospital, David Geffen School of Medicine at the University of California, Los Angeles, California 90095; and Protigen, Inc. (D.M.), Mountainview, California 94043

Address all correspondence and requests for reprints to: Buel D. Rodgers, Ph.D., Department of Animal Sciences, 124 ASLB, Washington State University, Pullman, Washington 99164-6351. E-mail: danrodgers{at}wsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-binding protein (IGFBP)-3 has intrinsic antiproliferative and proapoptotic functions that are independent of IGF binding and may involve nuclear localization. We determined that exogenous IGFBP-3 rapidly translocates to myoblast nuclei and that a 22-residue peptide containing the metal binding domain (MBD) and nuclear localization sequence (NLS) can similarly direct chimeric GFP into myoblast nuclei. Furthermore, a non-IGF-binding IGFBP-3 mutant inhibited myoblast proliferation without stimulating apoptosis. These results suggest that IGFBP-3 inhibits muscle cell growth in an IGF-independent manner that may be influenced by its rapid nuclear localization. We therefore identified IGFBP-3 interacting proteins by screening a rat L6 myoblast cDNA library using the yeast two-hybrid assay and two N-terminal deletion mutants as bait: BP3/231 (231 residues, L61 to K291) and BP3/111 (K181-K291). Proteins previously known to interact with IGFBP-3 as well as several novel proteins were identified, including RNA polymerase II binding subunit 3 (Rpb3). The domain necessary for Rpb3 binding was subsequently identified using different IGFBP-3 deletion mutants and was localized to the MBD/NLS epitope. Rpb3/IGFBP-3 binding was confirmed by coimmunoprecipitation assays with specific antisera, whereas a NLS mutant IGFBP-3 did not associate with Rpb3, suggesting that a functional NLS is required. Rpb3 facilitates recruitment of the polymerase complex to specific transcription factors and is necessary for the transactivation of many genes. Its association with IGFBP-3 provides a functional role for IGFBP-3 in the direct modulation of gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MULTIFACTORAL regulation of skeletal muscle growth and development is controlled extracellularly by many different growth factors and cytokines, including IGF-I and -II (1). The IGFs stimulate myoblast differentiation by modulating the intracellular levels and activity of cyclin-dependent kinases and other downstream effectors (2, 3), whereas they are also potent mitogens capable of stimulating myoblast proliferation via MAPK-dependent pathways (4). This unique ability to stimulate both aspects of muscle development is dependent upon cell cycle progression for myoblast proliferation and by contrast, growth arrest for differentiation; two opposing processes that in general are inversely regulated. Thus, myoblast proliferation and/or differentiation may be under autocrine control by IGF-regulated mediators that possibly include the IGF binding proteins (IGFBPs).

Members of this highly homologous protein family directly modulate IGF biological activity and bioavailability (5). In circulation, they help to maintain an IGF reservoir and increase ligand half-life, whereas at the cellular and tissue level, IGFBPs can either attenuate or augment IGF activity by moderating ligand/receptor interactions. Individual IGFBPs are differentially expressed in a variety of tissues and cell types, including skeletal muscle, and most if not all tissues are exposed to multiple IGFBPs from endocrine, autocrine, or paracrine sources. It is becoming increasingly clear, however, that some IGFBPs and in particular IGFBP-3 can influence cellular activities without interfering with IGF availability or receptor binding (5, 6). In addition, IGFBP-3 nuclear localization has been confirmed in some cell types and is dependent upon an intact nuclear localization sequence (NLS) (7, 8, 9, 10, 11). Liu et al. (10) demonstrated that direct interactions between IGFBP-3 and the nuclear retinoid X receptor (RXR) {alpha} enhanced the transcriptional activity of retinoid response elements and is essential to the apoptotic effects of IGFBP-3 in a prostate cancer cell line. Nevertheless, a definitive physiological role for IGFBP-3 in the nucleus has yet to be described.

Recent studies suggest that muscle cells secrete multiple IGFBPs and that IGFBP-3 is specifically involved in the growth regulation of these cells (12, 13, 14, 15, 16). In primary cell cultures composed of approximately 50% human myoblasts, IGFBP-3 secretion was proportional to the degree of differentiation and was up- and down-regulated by factors that either stimulate (IGF-I) or inhibit (TNF-{alpha}) this process, respectively (15). Furthermore, IGFBP-3 antisense oligonucleotides similarly reduced IGFBP-3 production and myoblast differentiation, suggesting that IGFBP-3 is a differentiation factor under these unique polyculture conditions. IGF-stimulated proliferation of porcine embryonic myogenic cells was inhibited by the addition of equimolar amounts of IGFBP-3 (14). Similar results were obtained when using the Long-R3 IGF-I analog that does not bind IGFBP-3, suggesting that the growth inhibitory effects of IGFBP-3 was not due to sequestering IGF-I. In a related study, the authors also demonstrated that growth inhibition by myostatin or TGFß1 is associated with increased IGFBP-3 production and that immunoneutralization of endogenous IGFBP-3 partially attenuates the actions of these cytokines (16). Taken together, these studies suggest that IGFBP-3 may inhibit IGF-stimulated myoblast proliferation by both IGF-dependent and -independent means, which in turn may influence differentiation.

Reported herein is the inhibition of basal myoblast proliferation by a non-IGF-binding IGFBP-3 analog (17). Using fluorescent and confocal microscopy and Western blotting of cytosolic and nuclear fractions, we have also shown that IGFBP-3 rapidly localizes to the nuclei of rat L6 myoblasts and that the MBD of IGFBP-3 is likely responsible for translocating across both plasma and nuclear membranes as well as associating with RNA polymerase binding subunit 3 (Rpb3). This domain is also necessary for the IGF-independent growth inhibitory effects of IGFBP-3 on myoblasts. Taken together, these results support an emerging model where IGF-stimulated myoblast differentiation is mediated in part by the local production of IGFBP-3, its rapid localization into the nucleus, and subsequent association with Rpb3 and the resulting inhibition of myoblast proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Stable transformation of Chinese hamster ovary (CHO) cells
The cDNA for a previously described non-IGF-binding human IGFBP-3 with glycine substitutions for residues critical to IGF-binding (I56, L80, and L81) was kindly provided by Ron Rosenfeld (17). The mutant cDNA was subcloned from pCMV6-(GGG)BP-3 into the pcDNA3.1 expression vector creating pcDNA3.1-(GGG)BP-3. CHO cells were transfected with 5 µg pcDNA3.1 or pcDNA3.1-(GGG)BP-3 and LipofectAmine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Stably transfected cells were derived in 600 µg/ml G418, and six monoclonal CHO/empty vector (EV) and CHO/(GGG)-IGFBP-3 lines were eventually expanded. The presence of mutant protein in the serum-free conditioned medium from three CHO/(GGG)IGFBP-3 clonal cell lines was verified by Western blotting with {alpha}hIGFBP-3 (Diagnostic Systems Laboratory, Webster, TX). In addition, serum free conditioned medium was collected from confluent CHO/(GGG)IGFBP-3 clone 1 cells after 48 h and the concentration of mutant protein was measured using a hIGFBP-3 enzyme-linked immunosorbant assay (Diagnostic Systems Laboratory).

Proliferation and apoptosis assays
Equal numbers of CHO/EV and CHO/(GGG)IGFBP-3 cells were grown to confluency in F-12K medium, which was then replaced with myoblast growth medium (DMEM/10% fetal bovine serum) for 24 h. This conditioned media were then used to replace the nonconditioned growth medium on myoblasts that were previously plated in 96-well plates and grown to approximately 50% confluency. Proliferation (CellTiter 96; Promega, Madison, WI) and apoptosis (caspase-3 and -7, ApoONE; Promega) assays were performed on these cells after 24 h according to the manufacturers’ protocols. Proliferation assays were also performed on L6 myoblasts (50% confluent) cultured in serum-free DMEM supplemented with 1 nM long R3 (LR3) IGF-I (Diagnostic Systems Laboratory) and 1.0 or 0.5 nM recombinant IGFBP-3 or the MDGEA IGFBP-3 that has a mutated NLS; 228KGRKR to 228MDGEA (18). Experiments were repeated twice (Fig. 1Go, n = 12/experiment; see Fig. 8Go, n = 7/experiment) and differences between groups were determined by a Student’s t test or by an ANOVA coupled to Fisher’s least significant difference test for multiple mean comparisons. Statistical analyses were performed on individual assays and on pooled data by expressing the arbitrary values as a percentage of controls (Fig. 1Go, n = 24; see Fig. 8Go, n = 14) and differences were detected using both approaches.


Figure 1
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  		FIG. 1. IGF-independent inhibition of myoblast proliferation by IGFBP-3. A, Western immunoblotting of conditioned media from three stably transfected CHO cell lines overexpressing a non-IGF-binding IGFBP-3 termed (GGG)BP-3. B, Detection of IGFBP-4 in conditioned media of CHO cells transfected with EV or with (GGG)IGFBP-3. Media from confluent cells were concentrated 10-fold by spin filtration and the end volumes were normalized. An equal volume from each medium was then analyzing by Western blotting with {alpha}IGFBP-4. C, Conditioned medium from CHO cells transfected with an empty vector or from CHO/(GGG)BP-3 cells was added to proliferating myoblasts for 24 h. Cell number and apoptosis assays were performed as described in Materials and Methods. The arbitrary values from both proliferation and apoptosis assays are expressed as a percentage of controls, CHO cell conditioned medium values (n = 24).

 

Figure 8
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  		FIG. 8. IGFBP-3 inhibition of myoblast proliferation requires an intact NLS domain. Proliferating L6 myoblasts were incubated for 48 h in serum-free DMEM with or without 1.0 nM LR3 IGF-I (does not recognize IGFBPs) in the presence of 1.0 and 0.5 nM wild-type IGFBP-3 (B) or the MDGEA mutant IGFBP-3 (M), which is incapable of nuclear localization (5 ). Cell number was determined using the CellTiter 96 colorimetric assay (Promega) according to the manufacturers’ protocol (mean values ± SEM are shown; n = 14/treatment).

 
Fluorescent microscopy
Rat L6 myoblasts were plated in DMEM supplemented with 10% fetal bovine serum and grown to 50% confluency on coverslips. Cells were then cultured in the absence or presence of 500 ng/ml recombinant human IGFBP-3 (Protigen, Inc., Mountainview, CA) for 60 min. After washing three times in PBS, cells were then fixed in 1% paraformaldehyde on ice for 15 min, washed three additional times, and permeabilized on ice with 0.2% Triton X-100 for 15 min. Nonspecific binding sites were blocked by incubating cells in 3% normal goat serum diluted in PBS for 1 h at room temperature. Cells were subsequently probed with {alpha}hIGFBP-3 (1:200; Diagnostic Systems Laboratory) for 1 h at room temperature, washed three times and then similarly incubated with a fluorescein isothiocyanate (FITC)-conjugated rabbit antigoat secondary (RAGFITC, 1:300; Vector Laboratories, Burlingame, CA) in the dark. Cell nuclei were labeled by staining DNA with 5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) for 2 min, also in the dark. Coverslips were then mounted with Fluoromount (Diagnostic BioSystems, Pleasanton, CA) antifading reagent and sealed. Positive IGFBP-3 immunoreactivity and DNA staining of cells treated with hIGFBP-3 and of controls—untreated cells stained with both primary and secondary antisera or treated cells stained with secondary alone—was detected using fluorescent microscopy. In separate experiments, immunolocalization of exogenous IGFBP-3 was also determined as described using cells that were treated for 5, 20, and 60 min. In these experiments, IGFBP-3 immunoreactivity was visualized using a two-photon Leica (Bannockburn, IL) TCS SP confocal microscope.

To determine whether the MBD region of IGFBP-3, which also contains the NLS and caveolin binding box, possibly facilitates plasma as well as nuclear membrane translocation in these cells, myoblasts were incubated with 500 ng/ml GFP-chimeric peptides (Protigen, Inc.) that corresponded to either the carboxy-terminal MBD/NLS region (242KKGFYKKKQCRPSKGRKRGFCW263) or to residues 176–194 of IGFBP-3 (KKGHAKDSQRYKVDESQS, control peptide). Cells were terminated after 5, 20, and 60 min, fixed and DNA was stained as described. Cellular localization of the chimeras was then visualized by confocal microscopy. These were among many recently characterized IGFBP-3 peptide-GFP chimeras, GFP32 and GFP31, respectively (19).

Cellular fractionization and Western blotting
Replicate cultures of L6 myoblasts were grown to approximately 70% confluency and treated with or without 500 ng/ml IGFBP-3 for 60 min. Myoblasts were also grown to 50% confluency and stimulated to differentiate by serum withdrawal; replacing growth medium with DMEM/2% horse serum. Myoblasts and fully differentiated myotubes were washed five times in PBS at room temperature and cytosolic and nuclear protein was isolated with the CelLytic NuCLEAR fractionation kit (Sigma, St. Louis, MO) according to the manufacturer’s protocol. Protein concentration was determined by the Lowry assay (Bio-Rad, Hercules, CA) and 30 µg from each replicated fraction were separated by denaturing PAGE under reducing conditions (10% ß-mercaptoethanol). Protein was transferred onto a 0.2 µm polyvinylidene difluoride membrane (Bio-Rad), which was subsequently blocked in 5% nonfat milk (Bio-Rad) prepared in 20 mM Tris.HCl (pH 7.5), 137 mM NaCl, and 0.1% Tween 20 (TBST). The membrane was then probed with {alpha}hIGFBP-3 (1:2000; Diagnostic Systems Laboratory) for 60 min in 5% milk, subjected to 3- to 10-min washes in TBST and reprobed with a RAG secondary for 30 min, all at room temperature. After repeating the washes, positive immunogenic reactions were visualized with enhanced chemiluminescence reagents in combination with Hyperfilm-ECL (both from Amersham Life Science, Arlington Heights, IL).

Yeast two-hybrid assays
Total RNA was isolated from L6 myoblasts with Trizol (Invitrogen) and mRNA was further isolated using oligo(dT) cellulose following the MicroPoly(A) Purist (Ambion, Austin, TX) protocol. Double-stranded cDNA was generated using Moloney murine leukemia virus reverse transcriptase and oligo(dT) primers with the BD SMART kit (BD Biosciences, Palo Alto, CA) and a cDNA library was constructed from PCR-amplified cDNA using the BD Matchmaker library construction and screening kit (BD Biosciences). Two separate bait vectors were constructed from the wild-type hIGFBP-3 cDNA, BP3/231 (231 amino acids, L61-K291) and BP3/111 (K181-K291), which were ultimately used in the screening assay. Flanking EcoRI and BamHI sites were introduced by PCR (25 cycles of 94 C for 30 sec, 60 C for 30 sec, and 68 C for 1 min) using primers listed in Table 1Go. The resulting amplicons were then subcloned into pGBKT7 in-frame with the GAL4 DNA binding domain producing pGBKT7-BP3/231 and -BP3/111. Before screening, yeast (AH109) were transformed with both plasmids separately to ensure that neither induced reporter gene transactivation alone. Yeast were then cotransformed with the GAL4 activation domain pGADT7-Rec vector, double stranded cDNA and pGBKT7-BP3/231 or –BP3/111 vectors and allowed to grow at 30 C on high-stringency synthetic dropout (SD) plates lacking tryptophan, leucine, adenine, and histidine (Trp–/ Leu–/ Ade–/ His–). Colonies were collected and cultured in 3 ml of liquid SD/-Trp/-Leu/-Ade/-His for 24 h. Cells were harvested, resuspended in 100 µl of SD/-Trp/-Leu/-Ade/-His medium supplemented with 100 U of Lyticase (Sigma) and incubated at 37 C for 1 h under vigorous shaking. Twenty microliters of 20% sodium dodecyl sulfate were added to each sample and tubes vortexed vigorously. Samples were put through a freeze/thaw cycle at –20 C and vortexed to ensure complete cell lysis. Both pGADT7 and pGBKT7 recombinant plasmids were then isolated using the QIAprep miniprep kit (QIAGEN, Valencia, CA) and transformed into DH5{alpha} Escherichia coli strain. pGADT7 library plasmids containing cDNA for IGFBP-3-interacting proteins were amplified and separated from pGBKT7 vectors by growing transformed DH5{alpha} in the presence of ampicillin alone. Different IGFBP-3 and Rpb3 deletion mutants were similarly constructed using PCR as described above (see Table 1Go) and used to confirm the library screening results as well as to identify the interacting domains. These include BP3/39 (P232-P270) subcloned into pGBKT7 and Rpb3/120 (M1-L120) and Rpb3/157 (R118-N275) into pGADT7. Such forced interactions were performed by first growing transformed yeast on Trp–/ Leu– plates and by restreaking colonies on Trp–/ Leu–/ Ade–/ His– plates.


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  		TABLE 1. Subcloning primers

 
Coimmunoprecipitation assays
Rpb3 (purchased under the commercial name of POLR2C; USBiological, Swampscott, MA) was incubated for 3 h on a rotary shaker at room temperature with either the recombinant IGFBP-3 or the MDGEA-IGFBP-3 mutant in 500 µl of the binding buffer (150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 and 0.5% CHAPS in 1x PBS) and at a concentration of 1 µg/ml each. Antibodies against either IGFBP-3 (rabbit anti-IGFBP-3 IgG; Santa Cruz Biotechnology, Santa Cruz, CA) or Rbp3 (chicken anti-POLR2C IgY; ProSci Inc., Poway, CA) were added at titers of 1:50 and 1:200, respectively, and the reactions incubated for an additional 1 h. When chicken anti-POLR2C antibodies were used, rabbit antichicken IgY antibodies (Immunology Consultants Laboratory, Newberg, OR) were added (1:200) for an additional 1 h incubation (IgY type antibodies are not recognized by proteins A or G). Protein/antibody complexes were precipitated using 50 µl of Protein G Plus/Protein A-Agarose beads (Calbiochem, San Diego, CA) prewashed with the binding buffer. Precipitated complexes were washed three times with fresh binding buffer, solubilized in Laemmli Loading buffer and boiled for 10 min. Fractions were loaded on a SDS-PAGE gel and immunoblotted for the presence of IGFBP-3, the MDGEA-IGFBP-3 mutant, or of Rpb3. Binding interactions between exogenous IGFBP-3 and endogenous Rpb3 were also investigated using nuclear lysates. Proliferating L6 myoblasts (~80% confluent) were incubated with or without 1 µg/ml IGFBP-3 for 60 min. Cells were washed thoroughly, and nuclear protein was extracted using the Nuclear Complex Co-IP Kit according to the manufacturer’s protocol (Active Motif, Carlsbad, CA). To preserve potentially unstable protein/protein interactions, enzymatic shearing was performed at 4 C for 90 min instead of the standard 10 min at 37 C. Nuclear protein was immunoprecipitated as described above using the manufacturer’s buffer and protocol with the exception of the following modifications: each reaction included 500 µg of nuclear protein mixed with or without 5 µg of polyclonal {alpha}IGFBP-3 (Santa Cruz Biotechnology) and both the incubation and the washing buffers were supplemented with 150 mM NaCl and 0.5% CHAPS, but did not contain dithiothreitol. Immunoprecipitated proteins were then detected by immunoblotting as described with either {alpha}IGFBP-3 or {alpha}Rpb3.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-independent inhibition of myoblast proliferation
The presence of mutant protein in the conditioned medium of three CHO/(GGG)IGFBP-3 cell lines was confirmed by Western blotting (Fig. 1AGo) and was additionally measured in the serum-free conditioned medium of the first clonal line by an ELISA (338.5 ng/ml). Conditioned myoblast growth media (DMEM/10% fetal bovine serum) from control CHO cells stably transfected with an empty vector (CHO/EV) or from CHO/(GGG)IGFBP-3 clone 1 were then used to determine the IGF-independent effects of IGFBP-3 on myoblast proliferation. The number of proliferating myoblasts cultured for 24 h with medium containing (GGG)IGFBP-3 was approximately 35% less than cells cultured with CHO/EV conditioned medium (Fig. 1CGo). Thus, IGFBP-3 directly inhibited myoblast proliferation in vitro without necessarily sequestering locally produced IGF-I or that contained within the serum. This effect was not due to plating efficiency or to apoptosis because caspase-3/7 activity was identical in both groups, suggesting that the (GGG)IGFBP-3-induced reduction in myoblast cell number was due to either a reduced proliferation rate or to cell cycle growth arrest, but not to cell death. CHO cells express predominantly IGFBP-4, which were determined to be equal in conditioned medium from both CHO/EV and CHO/(GGG)IGFBP-3 cells (Fig. 1BGo). Levels of IGFBP-4 were in fact identical in media from all CHO/(GGG)IGFBP-3 clonal cell lines despite sometimes very different levels of mutant IGFBP-3 (data not shown). Therefore, the overexpression of (GGG)IGFBP-3 does not result in a compensatory increase in IGFBP-4 secretion. These results additionally suggest that the growth inhibitory effects of the CHO/(GGG)IGFBP-3 medium was due to the mutant IGFBP-3 and not to changes in IGFBP-4 production.

Nuclear localization of recombinant IGFBP-3 in rat L6 myoblasts
IGFBP-3 immunoreactivity was absent in control cells incubated without recombinant protein and stained with both primary and secondary antisera (Fig. 2Go, middle panel) and was similarly lacking in control cells incubated with 500 ng/ml IGFBP-3, but stained with secondary antibody alone (Fig. 2Go, left panel). Thus, these primary and secondary antisera do not cross-react with nonspecific proteins under these conditions. In cells treated with recombinant protein and stained with both antisera, IGFBP-3 immunoreactivity was located within myoblast nuclei as indicated by overlapping staining of DNA (blue) and IGFBP-3 (green) (Fig. 2Go, right panel). These results suggest that exogenous IGFBP-3 translocates across the plasma and nuclear membranes of proliferating myoblasts in vitro and ultimately nuclear locates within the 60-min culture period. Cells were fixed in paraformaldehyde and washed extensively (six times total) before permeabilization. Therefore, nuclear localization of IGFBP-3 is not due to contamination of nuclear and other intracellular compartments during the immunolabeling procedure. Plasma membrane and/or cytosolic staining was also detected in these cells (right panel) but could not be distinguished from one another using normal fluorescent microscopy. Subsequent studies were, therefore, performed using a confocal microscope. Western blotting of equal amounts of cytosolic and nuclear protein from cells incubated with IGFBP-3 for 60 min independently verified the presence of IGFBP-3 in the nucleus (Fig. 2BGo). The protein was intact in both cellular fractions and was not proteolyzed. Thus, the in situ immunofluorescence of IGFBP-3 in the former assays is not due to immunoreactive byproducts of IGFBP-3 degradation. Absolute levels of IGFBP-3 were greater in nuclear protein fractions than in cytosolic, which is consistent with the transitional movement of IGFBP-3 from the cytosol to the nucleus. Intact IGFBP-3 was additionally detected in nuclear protein of fully differentiated myotubes as well as in proliferating myoblasts, which suggests that a nuclear role for IGFBP-3 is preserved even after differentiation.


Figure 2
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  		FIG. 2. IGFBP-3 nuclear localization in L6 myoblasts. A, Cells were incubated –/+ 500 ng/ml IGFBP-3 for 1 h, fixed and stained –/+ antihuman IGFBP-3 ({alpha}BP-3) and –/+ FITC-conjugated rabbit antigoat secondary (RAGFITC). Blue DAPI staining of nuclei (D) and green RAGFITC staining of IGFBP-3 (F) images are inset of overlay images. Colocalization/staining of DNA and IGFBP-3 is indicated by turquoise. Left and middle panels indicate no nonspecific staining of secondary and primary antisera, respectively. B, Western blotting of cellular fractions from L6 myoblasts and myotubes incubated with IGFBP-3. Myoblasts and fully differentiated myotubes were incubated –/+ 500 ng/ml IGFBP-3 for 60 min. Cytosolic (c) and nuclear (n) fractions were then blotted with {alpha}hIGFBP-3. BP-3, Short and long exposures of 20 ng IGFBP-3 peptide.

 
IGFBP-3 nuclear localization time-course
Immunolocalization of IGFBP-3 was determined in cells treated for 5 and 20 min (Fig. 3AGo). A minimal amount of exogenous IGFBP-3 was located within the nuclei after only 5 min as the majority of immunoreactivity was cytosolic and perinuclear. By contrast, both cytosolic and nuclear compartments were entirely engulfed with IGFBP-3 after just 20 min. This rapid pattern of translocation was mimicked by the GFP chimera containing the MBD/NLS peptide (Fig. 3BGo), but not by the control chimera (Fig. 3CGo). These results suggest that the MBD/NLS region of IGFBP-3 may facilitate the crossing of both plasma and nuclear membranes. They further suggest that a NLS/MBD peptide could similarly direct other proteins or large hydrophilic molecules across these membranes and into the cytosol and/or nuclei of myoblasts and possibly other cell types. The identification of nuclear IGFBP-3 using fluorescent microscopy, confocal microscopy, and Western blotting all suggest, moreover independently verify, that IGFBP-3 rapidly translocates to myoblast nuclei.


Figure 3
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  		FIG. 3. IGFBP-3 and MBD peptide time course for nuclear localization in L6 myoblasts. A, Confocal microscopy of myoblasts incubated with 500 ng/ml IGFBP-3 for 5 and 20 min. Control cells incubated in the absence of IGFBP-3 were negative (data not shown). B, Myoblasts incubated with 500 ng/ml of a GFP-chimeric peptide that corresponds to the MBD/NLS domain of IGFBP-3 (MBD/NLS-GFP, residues 242–263) or to (C) a control peptide (residues 176–194 of IGFBP-3). Images of DAPI-stained nuclei (D) and RAGFITC-stained IGFBP-3/GFP fluorescent (F) are inset of overlay images.

 
Identification of IGFBP-3-interacting proteins
A total of 57 clones (12 duplicates) were identified as putative IGFBP-3-interacting proteins after screening a custom L6 myoblast cDNA expression library using a high stringency yeast two-hybrid assay (Fig. 4Go) and two different IGFBP-3 deletion mutants as bait: BP3/231 and BP3/111 (see Fig. 5Go). Fifteen clones were isolated with BP3/231 and 42 with BP3/111, possibly indicating steric hindrance by the N-terminal region. Thirteen proteins have been previously shown to directly interact with IGFBP-3 (10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32), and three of these were also identified in our screens, including fibronectin (22), type I {alpha}-collagen (23) and IGFBP-3 itself (29), which independently validates our assay (Fig. 4Go). Several diverse functional classes were represented among the clones, although extracellular matrix proteins were best represented with 16 total clones (11 different proteins and five duplicates). Most interesting, however, was the identification of a nuclear pore protein as well as six different proteins involved in transcription including three clones of the rat homolog for RNA polymerase II (RNAPII) binding subunit 3 (Rpb3, a.k.a. subunit C, GenBank accession no. NP_001012491) and two clones of a structurally similar subunit of RNA polymerase I (NP_001008331).


Figure 4
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  		FIG. 4. Identification of IGFBP-3-interacting proteins in L6 myoblasts. A custom cDNA expression library was screened using a high stringency yeast two-hybrid assay (BD Biosciences) and two different IGFBP-3 deletion mutants as bait: BP3/231 and BP3/111 (see Fig. 5Go). A total of 57 clones were identified, 15 with BP3/231 and 42 with BP3/111. These include 12 duplicates as well as three previously identified IGFBP-3-interacting proteins (table). The functional distribution of each protein isolated with the indicated bait construct is shown in the histogram. cyto, Cytosolic; endo, endocrine; ECM, extracellular matrix; met, metabolic; sig, signaling; trans, transcription. Previously identified IGFBP-3-interacting proteins are shown in the table, including those also identified in our assays (bold). *, Method of detection = yeast 2-hybrid screening (Y2H), ligand affinity/chromatography (affi); #, functional class = ECM, endocrine or related (endo).

 

Figure 5
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  		FIG. 5. Binding interactions between IGFBP-3 and a RNA polymerase II subunit, Rpb3. RNA Polymerase II binding subunit 3 (Rpb3) and the homologous protein from the RNA Polymerase I complex (Pol-I) were identified in the Y2H screen, both with BP3/111. These interactions were confirmed by coexpressing the indicated proteins in Y2H assays. The putative Rpb3-binding domain within IGFBP-3 was also identified using these assays. Left, Cartoon of IGFBP-3 and Rpb3 deletion mutants. Functional domains of IGFBP-3 are shaded and annotated and each IGFBP-3 and Rpb3 mutant is named based on the total number of residues within each mutant (WT, wild type). Right, Yeast were cotransfected with plasmids containing the cDNA for the indicated mutants (IGFBP-3 in pGBKT7 DNA-binding vector; polymerases in pGADT7-Rec[2] activation domain vector) and spread upon leucine/tryptophan-deficient plates (Leu–/Trp–). Only cells containing both plasmids will grow under these conditions. Individual colonies were also cultured under restrictive conditions (Leu–/Trp–/His–/Ade– = histidine and adenine deficient as well) where growth requires the interaction between both proteins and the consequential expression of reporter genes. SV40 + p53 = positive control; Lamin C and SV40 = negative controls.

 
To confirm the binding interactions of BP3/231 and BP3/111 with Rpb3, yeast two-hybrid assays were performed using yeast cotransfected with either the full-length Rpb3 or the negative control activation domain vector, simian virus 40 (SV40), in combination with BP3/231, BP3/111 or with a negative control DNA-binding domain vector, Lamin C. With each transfection, the presence of both DNA-binding and activation domain vectors was verified by conditional growth on Leu–/Trp– plates (Fig. 5Go). Cells overexpressing either BP3/231 or BP3/111 and Rpb3 grew after replating on Leu– /Trp– /His– /Ade– plates, which confirmed Rpb3 binding to these regions of IGFBP-3. However, neither of the negative controls grew under these conditions. Similar results were also obtained using RNA polymerase 1–1 (Pol-I; Fig. 5Go), which is structurally similar to Rpb3. This suggests that the Rpb3/IGFBP-3 binding interaction is specific within the limitations of this assay. To help identify the interacting domains, assays were also performed on yeast cotransfected with a smaller construct that codes for the 39 amino acid epitope containing the MBD/NLS (BP3/39) in combination with constructs for either the first 120 amino acids of Rpb3 (Rpb3/120) or the last 157 (Rpb3/157). Conditional growth only occurred in cells overexpressing BP3/39 and Rpb3/120, indicating that binding occurs between the N-terminal half of Rpb3 and the region of IGFBP-3 that contains the MBD/NLS epitope.

NLS requirement for IGFBP-3/Rpb3 binding and IGFBP-3-stimulated growth inhibition
The direct association of IGFBP-3 with Rpb3 was also confirmed using coimmunoprecipitation assays and antisera specific for each recombinant protein. When coincubated, Western blotting revealed the presence of both proteins in the {alpha}IGFBP-3 and {alpha}Rpb3 immunoprecipitates (Fig. 6Go) and there was no indication of antibody cross-reactivity (left and middle panels, last lanes). By contrast, only a minimal amount of Rpb3 was detected within the {alpha}IGFBP-3 immunoprecipitates when Rpb3 was incubated with the MDGEA mutant instead of wild-type IGFBP-3 (right panel, lane 2 vs. 3). These data suggest that an intact NLS is required for significant IGFBP-3/Rpb3 binding. This interaction was also confirmed by coimmunoprecipitating nuclear lysates from cells treated exogenously with wild-type IGFBP-3 for 60 min (Fig. 7Go). Both IGFBP-3 and endogenous Rpb3 were detected in the precipitates indicating coassociation of these two proteins within nuclear lysates. The presence of Rpb3 was not due to nonspecific interactions with the antiserum or with the protein A/G agarose as precipitates from cells treated without IGFBP-3 or lysates incubated with agarose alone did not contain Rpb3 (Fig. 7Go, last two lanes in both panels).


Figure 6
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  		FIG. 6. IGFBP-3/Rpb3 coimmunoprecipitation requires an intact NLS domain. Recombinant IGFBP-3 or the NLS-mutant that does not nuclear locate [MDGEA, see Firth and Baxter (5 )] were incubated with recombinant Rpb3 and immunoprecipitated as described in Materials and Methods with either IGFBP-3 or Rpb3 antisera as indicated. Precipitated complexes were washed, solubilized in SDS-PAGE loading buffer, and immunoblotted with the indicated antisera. Antibody specificity was controlled by immunoprecipitating IGFBP-3 with {alpha}Rpb3 or vice versa (left and middle panels, last lanes).

 

Figure 7
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  		FIG. 7. IGFBP-3/Rpb3 coimmunoprecipitation from L6 myoblast nuclear lysates. Proliferating cells were treated with or without recombinant IGFBP-3 for 60 min and washed thoroughly, and nuclear lysates were isolated as described in Materials and Methods. Equal amounts of nuclear protein from each treatment group were immunoprecipitated with {alpha}IGFBP-3 as indicated and samples were blotted as in Fig. 6Go. Nonspecific binding of nuclear protein to the protein-A/G agarose or to {alpha}IGFBP-3 was controlled by the inclusion of samples lacking either IGFBP-3 antibodies or peptide.

 
Proliferation assays were also conducted on myoblasts cultured for 48 h with LR3 IGF-I and either IGFBP-3 or the MDGEA mutant. As expected, LR3 IGF-I alone increased cell growth by almost 50% compared with controls (Fig. 8Go). Both doses of IGFBP-3, however, attenuated these effects, which confirm previous studies indicating a growth inhibitory role IGFBP-3 in these cells (14, 15, 16, 33) as well as the data presented in Fig. 1Go. This inhibitory effect was lost when cells were treated with the MDGEA mutant IGFBP-3. Thus, IGF-independent growth inhibition of myoblast proliferation requires IGFBP-3 nuclear localization and possibly Rpb3 binding.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Incubating myoblasts with IGF-I has a dual effect on the cell cycle because the growth factor simultaneously stimulates proliferation and differentiation (1, 34). It also stimulates the synthesis and secretion of IGFBP-3 because cells begin to differentiate, a process that appears to be significantly influenced by IGFBP-3 (12, 13, 14, 15, 16). We have shown that an IGFBP-3 analog with no appreciable affinity for either IGF-I or -II can inhibit myoblast proliferation under growth conditions, which is the first step in the initiation of differentiation. Similar roles have been defined for IGFBP-3 in negatively regulating myoblast growth using primary human (15) and porcine (14, 16) myosatellite cells as well as rat L6 myoblasts (33) that incorporate both IGF-dependent and -independent mechanisms. Thus, IGF-stimulated myoblast differentiation appears to be mediated, at least in part, by the local production of IGFBP-3, which serves a similar role for TGFß1 and myostatin (16).

Myoblast nuclear localization of IGFBP-3 was independently demonstrated in separate experiments using fluorescent and confocal microscopy and by Western blotting of nuclear protein. The translocation of intact IGFBP-3 across both plasma and nuclear membranes was rapid and occurred within 5 min, which is consistent with a functional nuclear role for IGFBP-3 in these cells and suggests that nuclear immunoreactivity was not due to nonspecific endocytosis and subsequent proteolysis of IGFBP-3. The presence of IGFBP-3 within the nuclei of many different cell types in addition to rat myoblasts and myotubes (7, 8, 9, 10, 11) suggests that IGFBP-3 helps in regulating fundamental cellular processes. However, IGFBP-3 stimulates apoptosis in many of these cells but had no effect on the myoblasts described herein. This suggests that, although the mechanisms required for IGFBP-3 nuclear entry may be relatively common, its function is likely cell type specific and dependent upon the unique expression of intracellular and nuclear proteins. Some IGF-independent effects of IGFBP-3 are dependent upon its nuclear localization, which may include myoblast growth inhibition. Indeed, the MDGEA mutant IGFBP-3, which does not nuclear locate, is incapable of inhibiting LR3 IGF-stimulated proliferation (Fig. 8Go) and does not associate with Rpb3 in coimmunoprecipitation assays (Fig. 6Go). We have additionally demonstrated exogenous IGFBP-3 binding to endogenous Rpb3 in nuclear lysates (Fig. 7Go) and although our data have not yet definitively identified an IGFBP-3:Rpb3 complex within myoblast nuclei in vivo, they strongly suggest that the binding interaction occurs and that nuclear translocation of exogenous IGFBP-3 is associated with direct binding to endogenous Rpb3. These data further suggest that nuclear localization and possibly Rpb3 binding, two related yet possibly exclusive events, are both necessary prerequisites for the IGF-independent inhibition of myoblast growth. The NLS domain is necessary for a number or binding interactions including the association of IGFBP-3 with importin-ß (5, 18). However, the NLS is also a highly and negatively charged domain that when mutated, may alter the three-dimensional structure of the C-terminal region, which would explain its dependence for so many binding interactions.

The first intracellular protein demonstrated to directly bind IGFBP-3 was RXR{alpha} (10), although Schedlich et al. (32) recently demonstrated retinoic acid receptor (RAR) binding as well. Binding to RXR{alpha} enhanced transcriptional activity and apoptosis in the former study, whereas RAR binding specifically inhibited ligand activation and the formation of RXR:RAR heterodimers in the latter. Thus, a minimum role for IGFBP-3 in the nucleus appears to include the modulation of transactivation for some RXR- and RAR-responsive genes, which in turn would have different effects depending upon the cell type and the presence of different retinoid receptors. Gene expression requires recruitment of the RNAPII complex whose core enzyme is composed of 12 different subunits. Rpb3 is the third largest subunit and is located externally away from the core DNA binding domain (35). Assembly of the RNAPII complex is dependent upon Rpb3, although its functional role, as is currently known, is to facilitate recruitment of RNAPII to specific transcription factors, which in turn initiates gene transcription (36, 37, 38, 39). Thus, nuclear IGFBP-3 may not need to bind DNA directly to influence transcription because it could assist in RNAPII recruitment to transcription factor complexes, including those involving RXR and RAR. Rpb3 is ubiquitously expressed in all tissues; however, its levels are considerably higher in cardiac and smooth muscle than in any other tissue (40, 41), which is suggestive of an alternative and muscle-specific role. Indeed, Rpb3 binds myogenin and activating transcription factor (ATF) 4 and directly facilitates their transactivational activity while simultaneously stimulating myoblast differentiation (38, 39). Corbi et al. (42) recently demonstrated Rpb3 shuttling between cytoplasmic and nuclear compartments. Its association with HCR ({alpha}-helix coiled-coil rod homolog) in the cytoplasm prevents nuclear entry and myoblast differentiation. Thus, a functional role for IGFBP-3 in regulating myoblast differentiation could include the delivery of Rpb3 to nuclear targets.

Lee et al. (43) recently determined that cellular internalization of secreted IGFBP-3 is mediated by its binding to transferrin and to caveolin. Although internalization, nuclear entry, and the apoptotic effects of IGFBP-3 were all completely blocked by inhibitors of caveolae formation, a minimal amount of a non-transferrin-binding mutant IGBFP-3 (K228E/R230G) still localized to the nucleus. In a similar study, Singh et al. (19) characterized several peptides based on an IGFBP-3 epitope within its C-terminal domain and defined critical motifs within these GFP-chimeric peptides necessary for cellular internalization. Chimeras containing the transferrin- and partial caveolin-binding motifs all localized to the nucleus, whereas those that lacked both motifs (GFP34) or the partial caveolin-binding motif alone (GFP36) remained extracellular. Coimmunoprecipitation, cross-linking, and/or ligand blotting assays were used to demonstrate direct binding between IGFBP-3 or the MBD/NLS peptide with caveolin (19, 43) and either transferrin (43) or its receptor (19). Together, these studies suggest that IGFBP-3 internalization is mediated by transferrin and caveolin, both of which are expressed in skeletal muscle cells including L6 myoblasts (44, 45), and that caveolin-binding in particular is critical.

Previous studies with transformed skeletal muscle cell lines suggested that IGFBP-5 was the principle IGFBP secreted by muscle cells (46). By contrast, recent studies with primary skeletal muscle stem cells from human and porcine sources suggest that IGFBP-3 is specifically involved in the growth regulation of these cells (14, 15, 16, 33). We hypothesize that IGFBP-3 partially mediates IGF-stimulated myoblast differentiation by inhibiting cell growth and that nuclear localization of IGFBP-3 is critical to this process (Fig. 8Go). Future studies will further explore the ability of IGFBP-3 to initiate differentiation and to regulate gene expression via its association with Rpb3. They will also determine whether other proteins identified in our screening assay contribute to or enhance these effects. Additional studies will determine whether the MBD/NLS domain can be exploited as a novel targeting agent for protein/drug delivery to intracellular compartments of muscle cells. Nevertheless, these and other recent studies strongly suggest that skeletal muscle development is influenced by the IGF-independent actions of IGFBP-3 and that nuclear localization is required.


   Footnotes
 
These studies were supported by grants from the United States Department of Agriculture (2001-35206-10078) and the Washington State University Foundation to Buel D. Rodgers and from the National Institutes of Health (1R01AG20954 and P50CA92131) (to P.C.). We are particularly grateful for the pCMV6-(GGG)IGFBP-3 vector provided by Ron Rosenfeld and the MDGEA mutant IGFBP-3 peptide from Robert Baxter and Sue Firth.

Disclosure summary: all authors have nothing to declare.

First Published Online February 2, 2006

Abbreviations: CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHO, Chinese hamster ovary; DAPI, 4',6-diamidino-2-phenylindole; EV, empty vector; FITC, fluorescein isothiocyanate; IGFBP, IGF binding protein; LR3, long R3; MBD, metal binding domain; NLS, nuclear localization sequence; RAG, rabbit antigoat; RAR, retinoic acid receptor; RXR, retinoid X receptor; SD, synthetic dropout; SV40, simian virus 40.

Received October 6, 2005.

Accepted for publication January 24, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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