Differential Regulation of Mannose 6-Phosphate Receptors and Their ...

THEJOURNALOF BIOLOGICAL CHEMISTRY (01991 by The American Society for Biochemistry and Molecular Biolow, Inc

Vol. 266, No. 9, Issue of March 25, pp. 5534-5539,1991 Printed in U.S. A.

Differential Regulation of Mannose 6-Phosphate Receptors and Their Ligands duringthe Myogenic Development ofC2 Cells* (Received for publication, July 13, 1990)

Gyorgyi Szebenyi and Peter Rotwein$ From the Departments of Medicine and Genetics, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 631 10

The mammalian insulin-like growth factor II/cationindependentmannose 6-phosphate receptor (IGF-II/ CIMPR) mediates both targeting and endocytosis of mannose 6-phosphate-containing proteins and binds insulin-like growth factor I1 (IGF-11). The cation-dependent mannose 6-phosphate receptor (CDMPR) lacks an IGF-11-binding site and participates only in the intracellular trafficking of lysosomal enzymes. During terminal differentiation of the myogenic C2 cell line,there is an increase in cell surface expression of the IGF-II/CIMPR in parallel with a rise in secretion of IGF-I1 (Tollefsen, S. E., Sadow, J. L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 15431547). In this study we show that IGF-II/CIMPRmRNA increases by more than 10-fold during the initial 48 h of C2 muscle differentiation with kinetics similar to the rise in IGF-I1 mRNA. Comparable levels of both mRNAs are expressed in C2 myotubes and in primary cultures of fetal muscle. By contrast, no change is observed in CDMPR transcript abundance during differentiation, and only a small, transient increase is seen in the enzymatic activities and mRNA levels of several lysosomal enzymes. The differential regulation of the two mannose 6-phosphate receptors during muscle differentiation suggests that they may serve distinct functions in development.

for the IGF-II/CIMPRin sorting newly synthesized lysosomal enzymes has been demonstrated (3, 4), although cells that lack this receptor still target lysosomal enzymes because of the presence of a second, cation-dependent mannose 6-phosphate receptor (CDMPR) (5). The reason for the existence of two functionallysimilarreceptorsisnot known. The two mannose6-phosphatereceptorsare localized tothesame subcellular compartments butdiffer in theirligand specificity. Both receptors transport lysosomal enzymes from the transGolgi t o a prelysosomal compartment and also cycle to the cellsurface(2, 5, 6). Only theIGF-II/CIMPR, however, internalizes extracellular ligands and binds IGF-I1 (7, 8). In contrast to the established role of the IGF-II/CIMPR in lysosomal enzyme targeting, its function with regard to IGFI1 action is controversial (9), since not only are many of the effects of IGF-I1 ingrowth and development mediated through the IGF-I receptor (10-12), but in two nonmammalian vertebrates the CIMPR does not bind IGF-I1(13,14). In mammals, however, arolefor the IGF-II/CIMPR in transmembrane signaling triggered by IGF-I1 is supported by the following observations. In human myoblasts, the metabolic effects of IGF-I1are only partiallyinhibited by anti-IGF-I receptor antibodies ( E ) , a n d in K562 human erythroleukemia cells, which lacks the IGF-I receptor, IGF-I1 promotes cell replication (16). In addition, there is evidence linking the IGF-II/ CIMPR to several second messenger systems. Upon IGF-I1 binding, thereceptorhasbeen shown toactivate calcium channels (17), to stimulateinositol phosphate turnover (18), nucleotide-binding proteins (19). The mammalian insulin-like growth factor II/mannose 6- and to interact with guanine Muscle development has been a useful model system for phosphate receptor (IGF-II/CIMPR)’is a multifunctional protein with distinct high affinity binding sites for two classes examiningthe expression andactions of IGFsandtheir of ligands: mannose 6-phosphate containing lysosomal en- receptors (20, 21). We havepreviously reported(21)that zymes and growth factors, and IGF-I1 (1, 2). A critical role there is a coordinate increase in IGF-I1 mRNA and protein andinthe cell surfaceexpression of theIGF-II/CIMPR * This work was supported by Basil O’Connor Starter Research during the terminal myogenic differentiation of C2 cells, a Grant 5-639 from the March of Dimes Birth Defects Foundation and cell line originally derived from mouse skeletal muscle (22). by grants from the National Institutes of Health. Oligonucleotides We now show that the up-regulation of receptor number in were synthesized at the Washington University Protein Chemistry these cells is preceded by a rise in IGF-II/CIMPR mRNA Laboratory, under support of the Diabetes Research and Training Center (National Institutesof Health Grant DK20579). The costs of that temporallyparallels the increase in IGF-I1mRNA. Durpublication of this article were defrayed in part by the payment of ing the samedevelopmental period, we find no change in the page charges. This article must therefore be hereby marked “adverabundance of CDMPR mRNA and only a small, transient tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate increase intheenzymaticactivitiesandmRNA levels of this fact. The nucleotide sequence($ reported in this paper has been submitted several lysosomal enzymes. Our results demonstrate that the two mannose 6-phosphate receptors are regulated by different to the GenBankTM/EMBL DataBank with accession number(s) M58585 and M58586. mechanismsduring muscle differentiation, suggesting that j: To whom correspondence should be addressed Washington Uni- each may play a distinct role in developing muscle.

versity School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-1086; Fax: 314-362-7183. MATERIALSANDMETHODS ’ Theabbreviations used are:IGF,insulin-like growth factor; Cell Culture-The mouse C2 muscle cell line (22) was grown on CDMPR, cation-dependent mannose 6-phosphate receptor; CIMPR, 0.2% (w/v) gelatin-coated 150-mm tissue culture dishes in Dulbecco’s cation-independent mannose 6-phosphatereceptor; PCR, polymerase chain reaction;PNP, paranitrophenyl; Pipes, 1,4-piperazinediethane- modified Eagle’s medium supplemented with 10% newborn calf serum, 10% fetal bovine serum (GIBCO), antibiotics, and antifungal sulfonic acid kb, kilobase(s).

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Mannose 6-Phosphate Receptors in Muscle Differentiation agents at 37 "C in a humidified 5% C02,95% airatmosphere. Undifferentiated cells were harvestej at about 60% confluency. Differentiation was induced at 70-80% confluency by changing the medium to Dulbecco's modified Eagle'smedium supplemented with 2% horse serum (GIBCO). Primary muscle cell cultures were established from mouse limb buds of embryonic ages 17-19 days. The tissue was minced, and then cells were dissociated in 0.25% trypsin and 0.05%DNase I in Hanks' balanced salt solution (GIBCO) in the absence of CaZ+and M e . Cells were plated in Dulbecco'smodifiedEagle's medium supplemented with 5% newborn calf serum and 10% horse serum (GIBCO) on collagenized plates. Under these conditions, myotubes form within 4 days. Differentiated cells were maintained in medium plus 0.5 p M cytosine arabinoside for 4 additional days, until they were harvested and cellular RNA was isolated. RNA Isolation-Cells were washed three times with cold Earle's balanced salt solution, collected using a rubber policeman, pelleted, and stored frozen at -80 "C until all samples in a series were harvested. Total cellular RNA wasextracted by differential precipitation after homogenizing the cells in guanidinium thiocyanate (23). The integrity of each RNA sample was assessed after electrophoresis in formaldehyde-agarose gels by staining with ethidium bromide. The quantity of RNA was determined spectrophotometrically. Molecular Cloning-A fragment of the mouse IGF-II/CIMPR gene was isolated by plaque purification after screening a X Charon 28 library with the bovine CIMPR cDNA (24, 25) by standard methods (26). The DNA contained in one hybridizing X recombinant was digested with restriction enzymes, and a 377-nucleotide exon-containing RsaI fragment was subcloned into plasmid Bluescript/KS (pBs/ KS, Stratagene, La Jolla, CA) for use as a hybridization probe. A 238-nucleotide portion of the mouse CDMPR cDNA was cloned from liver RNA, using the polymerase chain reaction (PCR) (27). First strandcDNA wasprepared from total neonatal liver RNA with Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories). Oligonucleotide primers for reverse transcription, for second strand cDNA synthesis, and for PCR amplification were synthesized using phosphoramidite chemistry. The DNA sequences of these primers matched regions of identity between the human (28) and bovine (29) CDMPR cDNAs.DNA amplification employing Taq polymerase and a Perkin-ElemerCetus DNA thermal cycler (Perkin-ElmerCetus Instruments) was performed onfirst strand cDNA using the following parameters: 30 cycles of 1-min denaturation a t 94 "C, 2-min annealing at 58 "C, and 3-min extension a t 72 'C, followed by a 10-min final extension at 72 "C. The product of this reaction was reamplified with nested primers using the same parameters as above, except that the annealing temperature was 55 "C. Amplified DNA of the expected size was cloned into theHincII site of pBS/KS. Fragments of the mouse 8-hexosaminidase 8 chain, cathepsins L and B cDNAs, were isolated from adult liver RNA by PCR amplification using specific oligonucleotides derived from published sequences (30-32). Nucleotide SequenceAnalysis-Double-stranded plasmid DNA was sequenced by the dideoxy chain termination method (33) using a modified T7 polymerase (Sequenase, U. S. Biochemical Corp.). All sequences were confirmed on both DNA strands. Probe Preparation-Recombinant plasmids were linearized at convenient restriction sites within each inserted DNA fragment or inthe polylinker region of pBS/KS 5' to the cloned DNA (see Figs. 1, 2, and 7). RNA probes complementary to mRNA were synthesized by in vitro run-off transcription using T3 or T7 RNA polymerase and ['"PICTP, as previously described (34). The size and integrity of the probes were analyzed by autoradiography after electrophoresis through 8 M urea, 6%acrylamide gels. RNA Analysis-Solution hybridization-nuclease protection assays were performed as described previously (34). Total cellular RNA (12.5 pg) was incubated with individual RNA probes a t 45 "C in a 15-pl reaction volume containing 80% formamide (v/v), 8 mM Pipes, pH 6.7,80 mM NaCl, and 0.2 mM EDTA for 14-18 h. Samples then were digested with RNases A and T1, followed by treatment with proteinase K and phenol/chloroform a extraction. Probe fragments protected from nuclease digestion were visualized following electrophoresis through 8 M urea, 6% acrylamide gels by autoradiography a t -80 'C with Kodak XAR5 x-ray film and twoDu Pont Lightning Plus intensifying screens. Exposure times varied from 6 to 24h. RNA abundance was calculated after scanning laser densitometry, using an LKB Ultroscan (Pharmacia LKB Biotechnology Inc.). Several exposures of each gel were scanned, and only those giving responses

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in the linear range were used for analysis. RNA blots were prepared by standard methods (35). Totalor polyadenylated RNA (isolated using the Stratagene poly(A) quick mRNA purification kit) was electrophoresed in formaldehyde-agarose gels, then transferred to nitrocellulose by blotting. The RNA was immobilized on the membrane by UV cross-linking using a Stratalinker-2400 in the auto cross-link mode (1200 pJ, 30 s). Filters were prehybridized for a minimum of 2 h and then hybridized to RNA probes for 15 h at 60 "C in fresh hybridization solution. Both the prehydridization and hybridization solutions were composed of 50% freshly deionized formamide, 5 X SSC, 100 mM NaP04,pH 6.5,0.2% sodium dodecyl sulfate, 5 X Denhardt's solution, and 2 mM EDTA. High stringency washes were performed for 30 min at 68 "C using two changes of 0.1 X SSC and 0.1% sodium dodecyl sulfate. Autoradiography was performed as described above. Assay for Lysosomal Enzymatic Actiuity-Cells were harvested and stored as described above. Cell pellets were resuspended in 150-500 p1 of buffer containing 50 mM Tris-HC1, pH 7.4, 1 mMMgC12, 1 mM NaCl, and 0.75% Lubrol and were subjected to three 5-min cycles of freezing and thawingand 5 min of sonication in aBranson Ultrasonic Cleaner. Conditioned cell media were collected sequentially after 12 h of conditioning, centrifugation for 10 min at 5000 X g to remove cellular contaminants, and concentrated 20-25-fold using an Amicon P-10 Centriprep. Enzymatic activities of 8-hexosaminidase and 8-glucuronidase were measured in 50 pl of solubilized cell extract, and P-hexosaminidase activity was measured in 100 pl of concentrated medium, as described by Warren (36). Each reaction was performed in a total volume of 500 p1 in 100 mM sodium acetate, pH 4.6, with 1 mM paranitrophenyl (PNP) glycoside as the substrate (PNP-8-glucosaminide or PNP-8-glucuronide, respectively). After an incubation period of 1-3 h at 37 "C, an equal volume of 1 M Na2C03was added, and the absorbance was measured at 400 nm. Experimental values were compared to a standard curve that was constructed using 1-100 nM solutions of PNP in 500 pl of reaction buffer. After subtracting values obtained for substrate blanks, specific activities were calculated as ng of PNP produced/60 min/mg of protein of cell extract. Protein concentration was determined by using a modified Bradford assay (Bio-Rad) with lysozyme as a standard. RESULTS

Isolation of a Portion of the Mouse IGFIIICIMPR Gene and a Fragment of the Mouse CDMPR cDNA-X clones containing fragments of the IGF-II/CIMPR gene were isolated from a mouse X Charon 28 library after screeningwith bovine CIMPR cDNA(25). The cloned DNA was mappedwith restriction enzymes; fragments which contained exons were identified by hybridization to the bovine cDNA, subcloned into pBS/KS, andsequenced. Fig. lA illustrates the structure of a genomic DNA subclone that was transcribed in vitro to obtain mouse IGF-II/CIMPR probes. Fig. 1B shows the nucleotide and deduced amino acid sequence of this 165-nucleotide exon that encodes a portion of the extracellular domain of the IGF-II/CIMPR. As indicated in Fig. lC,the deduced amino acid sequence of this exon is 81% identical with the bovine and 83%with the human IGF-II/CIMPR, confirming the authenticity of the cloned DNA as part of the IGF-II/ CIMPR gene. RNA probes transcribed from this IGF-11/ CIMPRtemplate hybridized to a 9-kb mRNA species in mouse liver and C2 myotube polyadenylated RNA.ZThus the length of the mouse receptor mRNA is similar to thatseen in bovine and human tissues(24, 37). Fig. 2A illustratesthestructureand Fig. 2B shows the sequence of a 238-nucleotide fragment of the mouse CDMPR cDNA that was cloned from neonatal liver RNA by PCR, using oligonucleotide primers corresponding to conserved regions of the bovine (29) andhuman (28) CDMPRs.The deduced protein sequence, excluding theprimers used in cDNA cloning,is 96% identical withthe human and94% with the bovine CDMPR, as indicated in Fig. 2C. The length of G. Szebenyi and P. Rotwein, unpublished experiments.

Mannose 6-Phosphate Receptorsin Muscle Differentiation

5536 A.

C2 cells at various stages of their myogenic development. Proliferating myoblasts express IGF-II/CIMPR mRNA low at I levels. During differentiation, there is an increase in IGF-11/ T7- - - - IGF-II/CIMPR T3 CIMPR mRNA, reaching a peak at 48 h and remaining at 97 261 that level through 96 h. B. Previous studieshave shownthat IGF-I1 mRNA abundance also rises during C2 cell differentiation (21). To compare the 80 aCCaCtgCaCt~atggtctctggacagctaggtgattcctgggtgtgggtcatgtcaccaaattgtgtgctttcctgt kinetics of IGF-I1 mRNA accumulationwith that of the IGF160 ggctttgcatgtctagGTMGGATGGGAGAGGAGAGCCTGTGTTCACILGGT~GGTGGACTGCACCTAC~~cAcATG II/CIMPR during C2 cell development, the sameRNA samLysAspGlyArgGlyGluProValPheThrGlyGluValAspCysTh~TyrPh~Ph~Th~T~ ples were usedfor analysis of IGF-I1mRNA by nuclease 240 GGACACTAlVLTACGCFfGCATC~GAGM~~GACFfCCTCTGCGGGGC~T~TGGCMG~CGCTATGACCTGT protection assay. A comparison of Figs. 3 (left, top) and 4 pAspThrLysTyrAlaCysIleLysGluLysGluAspLeu~uCysGlyAlaIl~snGlyLysLysA~g~~AspLeuS 320 indicates that the rate of rise of both mRNAs is similar, CTGTGTTGGCTCGTCACTCAGgtattgcttgcctttccgaggacagcagtcaccgtgttcacaggagatgaccccaatgg erValLeuAlaArgHisSer demonstrating that there is coordinate activation of both tttggggaaacattttttaattgccagttgagtcatgacccccattcccgtctagcc ligand and receptor gene expression during muscle differentiation in this cell line. C. The expression of IGF-I1 and IGF-II/CIMPRgenes during Mouse : K D G R G O P V F T G E ~ Y P P T U D T K Y A C I K B K E D L L C G A I N G ~ ~ L S V ~ S differentiation is not limited to theC2 cell line. In myotubes Bovine : NN---A---------------------VH---A----VSD--O-F---A----Human : N----T-----------------E---V----------TD--------A-V--A formed from primary cultures of embryonic mouse muscle FIG. 1. Structure and sequence of the mouse IGF-II/CIMPR cells, comparable levels of both mRNAswere found, asshown probe. A , an exon (box)with the surrounding introns(solid line)was in Fig. 5. subcloned into the vector pBS/KS (dotted line). This plasmid was Expression of the Two Mannose 6-Phosphate Receptor Genes linearized with EcoRI at the 5' end of the inserted gene fragment, Is Regulated Differently during Myogenic Differentiation-To and RNA probes were transcribed using T3 RNApolymerase. B , nucleotide and deduced amino acid sequence. Introns are in lower determine if the up-regulation of the IGF-II/CIMPR during case, and the exon is in upper case letters. C, comparison of the C2 cell differentiation reflects a general increase in expression deduced amino acid sequences for this portion of the mouse, bovine, of proteins involved in targetinglysosomal enzymes,we measand human IGF-II/CIMPR genes. Residues identical with themouse ured CDMPR mRNAlevels in the same RNA samples. Fig. 3 sequence are indicated by dashes. (left, middle)shows the result of a nuclease protection experiment using the CDMPR probe depicted in Fig. 2A and total A. RNA extracted from differentiating C2 myoblasts. As illusEcoRI trated, there is no change in the steady-state level of CDMPR mRNA in proliferating or differentiating cells. Identical reI sults were obtained by Northern blot analysis.' Fig. 3 (right T7""CDMPR - T3 1 238 panel) graphically summarizesresults of mRNA measurements for both receptors obtained by using RNA from four E. different series of differentiating C2 cells. A 4-fold increase in IGF-II/CIMPR mRNAwas observed during the first24 h 80 TMCCCTGTGTCTGAGGAGU;AGGCAlVLGTCCAGGATTGCTTCTACCTCT~GAGATGGATAGCAGCCTGGCCTGTTCAC of muscle differentiation, and a 13-fold increment was seen AsnProValSerGluGluArgGlyLysValGl~A~pCy~Ph~~~LeuPh~Gl~M~tAspSerSerLeuAlaCysSerP 160 CAGAGGTCTCACACCTCAGTGTGGGCTCGATCTTACTTGTCACATTTGCATCATTGGTTGCTGT~ATATCATTGGGGGGT by 48 h. By contrast, CDMPR mRNA levels remained conroGluValSerHisLeuSerValGlySerIleLeuLeuValThrPheAlaSerLeuV~lAl~ValTy~Il~Il~GlyGly stant throughout the same period. Therefore, the two man240 TTCTTATACCAGCGACTGGTAGTGGGGGGCC~GGG~TGGAGCAGT~CCTCCTCTGGCCTTCTGGCAGGATCTTGG nose 6-phosphate receptors are regulated differently during PheLeuTyrGlnArgLeuValValGlyAlaLysGlyThrGluGlnPheProProL~uAl~Ph~T~pGl~A~~L~~ muscle development i n vitro. The Enzymatic Activities and mRNA Levels of Lysosomal C. Enzymes Increase Transiently during Myogenic DifferentiaHouse :NGLV(IDCFYLFEMDSSLACSPDVSHLSVGSILLVTPASLVAVYIIGGFLYQRLWGALGTEQFPP~FTQDL B~"~:""""~"-"~"""~.""~"~ tion-Although expression of the two mannose 6-phosphate Human :"""""""""""~""-""-""""""~""""""""""""~~~ receptors is regulated differently during C2 cell differentiaFIG. 2. Structure and sequence of the mouse CDMPR probe. levels A , a fragment of the mouse CDMPR cDNA (box) was cloned from tion, theincrease in IGF-II/CIMPR mRNA and protein a requirement for enhanced lysosomal enzyme might reflect liver RNA by PCR and inserted into pBS/KS (dotted line). Probes mobilization during the tissue remodeling that accompanies were transcribed in a 3' to 5' orientation by T3 RNA polymerase using plasmid DNA linearizedwith EcoRI as the template. B , nucleo- myogenesis. To investigate thisissue, the enzymatic activities tide and deduced amino acid sequences. Sequences included in the of two lysosomal enzymes, @-glucuronidase and @-hexosaminprimers used for PCR amplification areunderlined. C, comparison of idase, which are both transported by the mannose 6-phosthe deduced amino acid sequences, excluding the PCR primers, for the mouse, bovine, and human CDMPR proteins. Residues identical phate receptors (38),were measured in proliferating anddifferentiating C2 cells. As Fig. 6 and Table I illustrate, there is with the mouse sequence are indicated by dashes. onlya transient rise in the intracellular activity of either the C2 cell mRNA detected with this probe is 2.4kb', the enzyme during C2 cell differentiation. Also, as shown in Figs. 7 and 8, the abundance of @-hexosaminidase @ chain mRNA same size as described in tissues from other species (28, 29). IGF-IIICIMPR mRNAIncreases during Muscle Differentia- and of mRNAs encoding two lysosomal endoproteases, cation-To determine if the previously reported increase (21) thepsins B and L, increased by only 2-4-fold at a single time in abundance of the IGF-II/CIMPR on the cell surface of point during the sameperiod of terminal muscle differentiadifferentiating myoblastsreflects enhancedreceptor gene tion. Thus, enhanced IGF-II/CIMPR expression is not parof these expression, solution-hybridization nuclease protection assays alleled by a corresponding rise inthesynthesis were performed to measure changes in IGF-II/CIMPR mRNA lysosomal enzymes. The Accumulation of @-Hexosaminidase i n C2 Cell-condicontent during the terminal differentiation of the myogenic C2 cell line. Fig. 3 (left, top) shows the result of a represent- tioned Medium Decreases during Muscle Differentiation-As a functional assayfor the mannose 6-phosphate receptors, the ative experiment in which the IGF-II/CIMPR probeillustrated in Fig. 1A was incubated with total RNA isolatedfrom accumulation of @-hexosaminidase activity in the cell media EcoRI

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