Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation

Cell, Vol. 79, 221-231,

October 21, 1994, Copyright

0 1994 by Cell Press

Muscle LIM Protein, a Novel Essential Regulator of Myogenesis, Promotes Myogenic Differentiation Siivia Arber,’ Georg Haider,t ‘Friedrich Miescher institute CH-4602 Base1 Switzerland tBiocenter University of Base1 CH4056 Base1 Switzerland

and Pica Carom’

Summary Muscle LIM protein (MLP) is a novel positive regulator of myogenesis. Its expression and that of its Drosophila homolog DMLPl are enriched in striated muscle and coincide with myogenic differentiation. in the absence of MLP, induced C2 ceils express myogenin but fail to exit from the cell cycle and to differentiate. Overexpression of MLP in C2 myobiasts potentiates myogenie differentiation and reduces its sensitivity to TGFf3. Like MLP, single LIM domain deletion mutants of MLP and nonmuscle LIM-oniy proteins promote myogenic differentiation. in 3T3 fibrobiasts, the same LIM proteins prevent phorboi ester-induced inhibition of DNA replication. These results establish MLP as an essential promoter of myogenesis and suggest that LlMoniy proteins act via similar mechanisms to regulate aspects of ceil differentiation. Introduction The formation of skeletal muscle during development is a multistep process that involves the determination of multipotential mesodermai ceils to give rise to myoblasts, withdrawal of the myobiasts from the cell cycle and differentiation into muscle ceils, and finally growth and maturation of skeletal muscle fibers. These processes are controlled by ubiquitous and muscle-specific transcriptional regulators that determine ceil fate and differentiation and by external signals that couple myogenesis to deveiopment and growth of the organism (Florini et al., 1991; Olson, 1992; Lyons and Buckingham, 1992; Sassoon, 1993). At the molecular level, myogenic determination and muscle-specific gene expression involve skeletal musciespecific helix-loop-helix (HLH) proteins and the myocyte enhancer-binding factor MEFP. The myogenic HLH proteinsor MyoD family members (MDFs) include MyoD, myogenin, Myf-5, and MRF4. Expression of MDFs in several nonmyogenic ceil lines is sufficient to induce competence for myogenic differentiation (for reviews see Lassar and Weintraub, 1992; Edmondson and Olson, 1993). MDFs act within a network of ubiquitous regulators of gene expression that affect central pathways of cell growth and differentiation. Among these are the inhibitor of MDFs id (Benezra et al., 1990) the immediate early gene products

Fos and Jun (Li et al., 1992; Bengal et al., 1992) and the tumor suppressor retinobiastoma protein (Gu et al., 1993). Several lines of evidence indicate that other (still unidentified) cofactors play important roles in qualitative and quantitative aspects of myogenesis and muscle-specific gene expression. Thus, detailed molecular studies on the mechanisms involved in the activation of muscle-specific genes by MDFs have implicated the probable involvement of coregulator proteins. These would play an essential role in muscle-specific activation of gene expression by MDFs (Weintraub et al., 1991; Schwarz et al., 1992). in addition, regulation of muscle maturation and growth during development, in the adult, and upon lesion probably involves factors that modulate the myogenic process. Such cofactors would control quantitative aspects of muscle-specific gene expression and mediate regulation of these processes by external signals. Here we report on a novel positive regulator of myogenesis. Owing to the presence of two LIM finger structures in its sequence, its pattern of expression, and its role in myogenic differentiation, we have called this protein MLP (for muscle LIM protein). MLP was isolated from a subtracted complementary DNA (cDNA) library enriched in genes induced in skeletal muscle by denervation. Based on its association with myogenic differentiation in vivo and in vitro and on the results of suppression and overexpression experiments in myogenic ceil lines, our findings indicate that MLP is an essential positive regulator of the myogenie program in determined myogenic cells and suggest that it may be a cofactor regulating muscle-specific gene expression in skeletal and heart muscle. Results A Novel Conserved LIM Finger Protein Expressed in Differentiating Striated Muscle Ceils and UpRegulated by Denervation To identify genes involved in the regulation of muscle gene expression and the formation of neuromuscular synapses, we have used a subtractive library approach to isolate cDNAs induced in rat skeletal muscle 7 days after denervation. As expected, most muscle messenger RNAs (mRNAs) wereeither unaffectedorslightiydown-regulated,andonly a small proportion of mRNAs was highly enriched in the denervated muscle. One of these cDNAs coded for a novel LIM finger protein (Figure 1) that we called MLP. The abbreviation LIM is derived from a common protein motif shared by the homeodomain proteins Lin-1 1, 191-1,and Met-3 (for an overview see Wang et al., 1992). The zincbinding LIM finger motif (Micheisen et al., 1993) consists of the sequence CxxCxl~IaC/HxxCxxCxl~lBC~~C (Figure 1D). The majority of LIM finger proteins have homeodomains, but exceptions include Cys-rich protein (CRP), the single LIM finger Cys-rich intestinal protein, the T cell oncogene rhombotin-1 , the focal adhesion protein zyxin, and

E

w

CRPorKP

ILY -4 Arm

GRIP or DMLPl

Isw

b Huu)-

j

uu

h

ESPl Rhombotin

Figure 1. Primary Structure of MLP and Its Relation to Other LIM Finger Proteins (A) Nucleotide sequence and predicted amino acid sequence of rat MLP. The open reading frame codes for a protein of 194 amino acids (24 kDa). (6) Nucleotide sequence and predicted amino acid sequence of a Drosophila homolog of MLP (DMLP7). The open reading frame codee for a protein of 92 amino acids with one LIM double finger. (C) Sequence alignment among chick MLP. chick CRP (Crawford et al., 1994). and DMLPI. In the sequences, underlined or star-marked residues contribute to the LIM finger structures. (0) Schematic representation of the zinc-binding LIM Gly-rich domain. (E) Schematic representation of the structure of known LIM finger proteins. GRIP, Cys-rich intestinal protein; ESPl, estradiol-stimulated protein 1; LIM, homeodomain LIM proteins.

kinase (Boehm et al., 1990; Wang et al., 1992; Sadler et al., 1992; Mizuno et al., 1994) (Figure 1 E). Analysis of the primary structure of MLP reveals the presence of two LIM fingers, each followed by a Gly-rich and hydrgphobic residue-rich region. This arrangement of the LIM finger region is common to a subset of LIM finger proteins (Figure 1 E). Among these, MLP is most homologous to CRP, an early response gene of fibroblasts that is expressed at high levels in the lung, but not in skeletal or heart muscle (Wang et al., 1992). MLP is a consenred protein: complementary RNA (cRNA) probes against rat MLP strongly hybridized with an mRNA of similar size in chick and Drosophila(Figure 28). Isolation

a novel protein

of chick A&P cDNA indicated that the protein is highly homologous to rat MLP (93% identity at the protein level) and less homologous to the related chick CRP (see Figure lC), confirming that MLP and CRP are distinct gene products. Hybridization of mouse genomic DNA with rat A&P cDNA at medium stringency revealed a simple pattern of hybridizing bands (Figure 2D), and results from a preliminary analysis of mouse genomic MLP clones are consistent with the possibility that the species detected on the blot all originate from the same lwLP gene (data not shown). Screening of a 12-24 hr Drosophila embryo cDNA library with rat A&P cDNA at low stringency yielded a Drosophila homolog of MLP (DA&PI). The cDNA codes for a

Muscle LIM Protein Promotes Myogenic Differentiation 223

A Ii

M

I

S

LU

LI

KI

B

P DO Do.6 DZ 03 D7 P3 P8

D

B

,

C

R

Z

9.

5

6

7

0

910

Dr

C od

Id

2d

54

Figure 2. Distribution of MLPand DMLPI mRNA: Enrichment in Striated Muscle, Induction in Muscte at the Onset of Dllerentiation, and Reinduction by Denervation in Adult Muscle (A) MLP mRNA in adult rat tissues. Abbreviations: H, heart; I, intestine; M, skeletal muscle; S, skin; LU, lung; LI, liver; KI, kidney; 8, brain; P, placenta. Denelvation (lower hindllmb muscle) is shown as days after denenration (Dn). Paralysis (gluteus muscle) is shown as days after Botulinum toxin A applkation (Pn). Scale bars show 28s and 18s rRNA. Rat MLP mRNA is approximately 0.9 kb. (B) Evolutionary conservation of MLP; hybridization with rat MLP probe. Lanes: R, 1 ng of adult rat skeletal muscle total RNA; C, 2 pg of chick El9 skeletal muscle total RNA; Dr, 5 ug of adult Drosophila melanogaster poly(A)+ RNA. A putative presplicing species like that detected in rat heart (shown in [A]) was the only additional hybridizing band detectable on these Northern blots. (C) Induction of MLP mRNA during C2 cell differentiation in vitro. d, days in differentiation medium. (0) Genomic Southern blot. Mouse DNA hybridized with the rat cDNA sequence shown in Figure IA. Lane 1, Sacl; lane 2, Pstl; lane 3, Notl; lane 4, Kpnl; lane 5, Hindlll; lane 6, EcoRV; lane 7, EcoRI; lane 9, Clal; lane 9, Bglll; lane 10, BamHI. Molecular weight markers are 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. (E) Localization of DAMP7 transcript during Drosophila embryogenesis. All embryos are oriented with anterior to the left. (a) Lateral view of an embryo at the end of germband retraction (early stage 13), when the first DMLPl transcripts can be detected. The visceral mesoderm and cells of the somatic mesoderm start to express DMLPl. (b) Late stage 13 embryo, ventral view. DMLPI transcripts are now readily detected in the visceral mesoderm (arrows) surrounding the endoderm and in a segmentally repeated pattern corresponding to somatic mesodermal cells underlying the ectoderm (arrowheads). (c) Stage 17 embryo, lateral view, superficial focat plane. All body wall muscles express DMLPI.

protein of 92 amino acids with one LIM Gly-rich motif (see Figure 16). The corresponding gene is located on chromosome 2, position 608. DMLPl aligns best to the first LIM Gly-rich domain of MLP (59% identity). The end of the DMLPl ‘coding sequence corresponds to the transition to the linker sequence that connects the two LIM Gly-rich domains in MLP and CRP. The expression of MLP was enriched in striated muscle (Figure 2A; see Figure 3E), where the mRNA was exclusively expressed by the muscle cells (in situ hybridization data not shown). In adult rat, it was highest in heart and was strongly up-regulated in denervated or paralyzed skel-

etal muscle (Figure 2A). A similar expression pattern was found for chick MLP. Finally, in the myogenic cell line C2, the onset of MLP expression coincided with differentiation (Figure 2C). The expression pattern of DMLPl during Drosophila embryogenesis was analyzed by in situ hybridization to whole-mount embryos. DMLP7 transcripts were first detected at the end of germband retraction (early stage 13, according to CamposOtega and Hartenstein, 1985) in the visceral mesoderm overlaying the endoderm and in ventral somatic mesodermal cells (Figure 2Ea). Signal levels had increased by the end of stage 13, and DMLPl transcripts were now readily detectable in the visceral

Cdl 224

Figure 3. Nuclear Accumulation

of MLP in Differentiating

Skeletal Muscle Cells

(A) Doublelabeling immunocytochemistry of cultured El 1 chick muscle cells. Antibodies are against MLP (a-d), myomesin (a’), a-actinin (b’), MHC (c’), and nuclei using DAPI (d). Times in culture were 2 days (a), 3 days (b), 5 days (c), and 7 days (d). (6) Detection of MLP (arrows) on immunoblots. Homogenate protein (70 pg) was from chick (CH) (El6 heart) or adult rat (control [M] and a-day denervated [D] skeletal muscle or heart [H]). Molecular size markers are 23, 31, 45, 99, and 97.4 kDa. (C) Contents of MLP mFfNA in embryonic chick hind limb. (D) Detection of MLP in C2 myotubes (4 days in differentiation medium). Double-labeling experiments were for MLP (a) and muscle MHC (a’). The insert shows a parallel culture stained with preimmune serum and double labeled for MHC. (E) MLP immunoreactivity on cryostat sections of adult rat tissues. MLP antibody (b-e) or the corresponding preimmune serum (a) was applied to sections of heart (a and b), placenta (c), skin (d), and brain (e). All sections were processed and photographed in the same way. Strong specific staining of hearf muscle cells is detected in (b); weak labeling of myometrium is detected in (c); very weak labeling of innermost structures in skin (d) and of the nuclei in some large hippocampal cells, presumably neurons (e), could also be detected with this antiserum. Scale bar: 60 pm (A and D) and 350 pm (E).

mesoderm and in a segmentally repeated pattern corresponding to somatic mesodermal cells (Figure 2Eb), where expression was now also detectable in more dorsally located cells. At later stages, strong accumulation of DA&P7 transcript was detected in all differentiating visceral and body wall muscles (Figure 2Ec). No signal was detected in the endoderm, in the ectoderm, or in the nervous system. Therefore, during Drosophila embryogenesis, DA&P7 expression is associated with developing muscles of the visceral and somatic mesoderm subsequent to the formation of muscle precursor cells.

When combined with the expression pattern of MLP in rat, chick, and myogenic cell lines, these findings indicate that the expression of MY in differentiating striated muscle cells is conserved during evolution, suggesting that the protein may play an important role in the differentiation and growth of striated muscle. MLP Accumulates in the Nucleus of Differentiating Myotubes To detect MLP in the absence of possible cross-reactivity with CRP, we produced an antiserum to the unique C-ter-

A

MLP

NE0

AS-MLP

MHC expression level

I

% of total in C2

-

II

% of total in each cell line

++

280

Figure 4. MLP Is an Essential

Positive Regulator

of Myogenic

Differentiation

in C2 Cells

(A) Stably transfected C2 cells after 3 days in G413containing differentiation medium. Transgene constructs are C2-neo (a), CBMLP (b), and CP-AS-MLP (c). Cultures were started at 25% confluency 1 day before transfer to differentiation medium. Inserts show parallel cultures after 3 days in G41Efree differentiation medium. In addition, these cultures were started at 25% confluency 2 days before transfer to differentiation medium. (8) Extent of myogenic differentiation in CS-MLP, CP-neo, and CPAS-MLP 3day cultures. High (+++), intermediate (++), and low (+) levels of MHC are as defined in Experimental Procedures. Values are from representative cultures (n = 5; standard error, approximately 10%). (C) MHC immunocytochemistry of CP-MLP (a) and CP-neo (b) cells (3 days in differentiation medium). (0) Rescue of myogenic differentiation in C2-AS-MLP cells transiently transfected with an MLP construct after 3 days in differentiation medium: insert-free plasmid (a and a’); MLP plasmid (b and b’); double labeling for MLP (a and b) and MHC (a’ and b?. (E) Quantitative analysis of rescue experiments as those shown in (D). Three independent control (NEO) and MLP transfections were analyzed. All MHC-positive cells were included in the analysis. Conversion efficiency was defined as the fraction of MLP-positive cells that also expressed detectable levels of MHC. The asterisk indicates that most cells expressing very high levels of the transgene (lo%-3W% of total) displayed immunoreactivity along actin filaments, but not in the nucleus, and did not express MHC (data not shown). Scale bar: 140 urn (A), 50 urn (C), and 100 pm (D).

minal peptide GGLTHQVEKKE. This antiserum specifically detects the corresponding protein in rat and chick muscle homogenates (Figure 36). Staining of cryostat sections of adult rat tissues yielded results consistent with thoseof the Northern blot data(Figure3E). in immunocytochemistry experiments with cultured cells, the antiserum detected a nuclear antigen in 2-day cultures from embryonicdayll (El l)chickmuscle(Figure3A). Inmoremature myotubes, MLP was not exclusively localized to the nucleus (Figures 3Ab and 3Ac). Absence of nuclear staining in some of these more mature myotubes was not due to overfixation, as variation of fixation time did not increase the fraction of stained nuclei (data not shown). When MLP immunoreactivity was localized in C2 myotubes, a pattern analogous to that detected in cultured chick myotubes was detected (Figure 3D). Therefore, like its mRNA, MLP protein is first detected during myotube formation, and its expression is particularly elevated during muscle maturation. In addition, in all differentiating myogenic cells analyzed in this study, the localization of MLP is initially exclusively nuclear. Later on, in maturing myotubes and muscle fibers, the protein also accumulates in the cytosol. MLP Is an Essential Positive Regulator of Myogenic Differentiation To explore the possibility that MLP may play a role in myogenie differentiation, we carried out both overexpression and antisense experiments in myogenic cell lines. No myogenie differentiation was detected when stably transfected C2 myoblasts that overexpressed MLP (CP-MLP) were grown in the presence of high serum. However, upon transfer to low serum conditions, CFMLP cells differentiated with markedly higher efficiency than cells transfer&d with an insert-free plasmid (CP-neo) or untransfected cells (Figure 4A). Stimulation of myogenic differentiation in C2MLP cells was detectable at the level of myotube frequency and size (Figure 4A), frequency and levels of expression of skeletal myosin immunoreactivity (Figure 48) total levels of skeletal myosin, MLP or myomesin (Figure 58) and expression of mRNAs characteristic of the myogenie phenotype, including an overproportional induction of MLP expression (Figure 5A). Differentiating C2-MLP cells had unusually large myotube diameters (see Figure 4C), suggesting that MLP may affect the level of expression or assembly of myofibril components in these cells. While overexpression of A&P in differentiating myoblasts promoted myogenic differentiation, suppression of mRNA expression prevented this process. When placed in differentiation medium, C2 ceils that expressed an MLP construct of the gene in an antisense orientation (CPASMLP) failed to form myotubes (see Figure 4A), expressed skeletal myosin at a very low frequency and at unusually low levels (see Figures 46 and 58) and contained essentially no detectable A&P mRNA (Figure 5A) or protein (Figure 58). Myogenic differentiation could be rescued in C2AS-MLP cells by transient overexpression of MLP (see Figures 4D and 4E), which led to MLP expression in a significant fraction of CP-AS-MLP cells (compare Figure 4Da with 4Db) and to skeletal myosin heavy chain (MHC) expression in about a third of these cells (Figures 4D and

4E). We noted, however, that cells producing the highest levels of MLP in these transient transfection experiments consistently failed to differentiate (data not shown). These cells displayed strong actin-associated and (in most cases) no nuclear MLP immunoreactivity. Conceivably, very high levels of MLP may be toxic or may interfere with differentiation in these cells. We obtained similar rescue results when bacterially expressed purified MLP was microinjected into C2ASMLP cells (data not shown). In conclusion, these results strongly suggest that the presence of MLP is essential for the expression of the myogenic phenotype in differentiating myotubes. The Requirement of MLP for Myogenesis Is First Detectable at the Exit from the Cell Cycle A role for hypothetical cofactors in myogenic processes has been postulated to allow and modulate musclespecific gene expression. Accordingly, their effects would first become apparent when differentiating myoblasts withdraw from the cell cycle (Nguyen et al., 1983). Owing to its first expression after myoblasts are transferred to differentiation-promoting conditions and its role in myogenesis, MLP may qualify as such a cofactor. To explore this possibility, we determined which steps in myogenesis are affected by MLP. Overproduction of MDFs in several nonmyogenic cell lines leads to the expression of muscle-specific genes upon transfer to differentiation-promoting conditions. To determine whether MLP has a similar activity, we generated C3HlOTl/2 (lOT1/2) lines that stably expressed MLP (1 OT-MLP). However, although lOT1/2 ceils are particulady responsive to MDFs, no muscle myosin immunoreactivity could be detected in 1 OT-MLP cells when these were maintained in differentiation-promoting conditions (data not shown). Therefore, unlike MDFs, MLP does not promote myogenic determination. In addition, although it consistently enhanced the contents of MHC-expressing cells, MLP also did not induce multinudeated myotube formation upon transient transfectlon in rhabdomyosamoma (RD) ceils (data not shown). Early differentiation events preceding exit from the cell cycle apparently did not depend on MLP. Thus, upon transfer to low serum-containing medium, myogenin induction was normal in the absence (C2-AS-MLP celts) or in the presence of excess (CS-MLP cells) MLP (Figure 5A). Similarly, induction of clone 4, a novel early marker of differentiation in myogenic cells (S. A. and P. C., unpub lished data), was not affected by the levels of MLP (Figure 5A). Finally, a further reaction of CP-ASMLP cells to differentiation medium was the induction of skeletal a-actinin (Figure 5D). This protein could not be detected in myoblasts. In contrast with the apparent lack of sensitivity of some early myogenic differentiation reactions to the presence of MLP, a drastic effect on the reduction of mitotic activity was detected when C2-AS-MLP cells were transferred to low serum medium (Figure 5C). The failure of CP-ASMLP cells to exit efficiently from the cell cycle in the presence of differentiation-promoting conditions was also evident when cell densities were compared in the cultures shown

Muscle LIM Protein Promotes 227

Myogenic

Differentiation

A AS-MLP ---

MLP

C2

012301230123

MLP

CI

w

*.a

m II I)-

Myogenin

MCK

B -

MLP AS

S

N “*r4

Myomesin

MHC AS

S

N

-

AS

S

N

rl

% 8rdU positive

C2 100% (49)

bFGF 50 nghnl

CZ-MLP 350% (17)

ND

Figure 5. The Dependence of C2 Differentiation tected at the Exit from the Cell Cycle

66%

(77)

on MLP Is First De-

(A) Contents of myogenic mRNAs in CP-neo, CP-MLP, and CP-AS-MLP cultures as a function of time (shown in days) in differentiation medium. (B) Contents of MLP, MHC, and myomesin (arrows) in C2-AS-MLP (AS), CP-MLP (S), and CP-neo (N) after 3 days in differentiation medium. (C) Percentage of DNA-replicating cells (BrdU incorporation) as a function of time (left column) in differentiation (Diff.) medium. Values from

in Figure 4A. C2AS-MLP cells did, however, not form foci, suggesting that they were not transformed (data not shown). To define further how MLP potentiates myogenic processes, we determined the sensitivity of CP-MLP cells to inhibitorsof myogenicdifferentiation. Myogenesis is under the control of growth factor signals from the environment, and it is potently inhibited by transforming growth factor pl (TGFpl) and basic fibroblast growth factor (bFGF) (Olson, 1992; Lassar and Weintraub, 1992; Edmondson and Olson, 1993). These growth factors prevent myogenic differentiation through different mechanisms that converge at the DNA-binding region of MDFs. Significantly, inhibition by TGFPl is restricted to the activation of musclespecific genes by MDFs, and it does not interfere with the activity of general transactivators of the E2A gene family (Martin et al., 1992). As shown in Figure 5E, overexpression of MLP in C2 cells had a marked effect on the inhibition of differentiation by TGFPl. In the presence of inhibitory growth factors, C2-MLP cells differentiated more efficiently than control cells. In addition, while strongly affected by bFGF, CP-MLP cells were markedly less sensitive toTGFP1 than control cells (Figure5E). These findings are consistent with the conclusion that MLP efficiently potentiates myogenic differentiation in C2 cells and suggest that it may regulate processes controlling muscle-specific gene expression. Like MLP, Single LIM Finger Deletion Constructs of MLP and Nonmuscle LIB&Only Proteins Can Promote Differentiation in Myogenic Cells and Confer Resistance to TPA-Induced Suppression of Proliferation in Fibroblasts Close examination of the primary structure of MLP suggests that the two LIM Gly-rich motifs may be the only functional domains of this protein. Their striking similarity suggests that they may function independently and similarly. Further plausibility for this possibility derives from the fact that DMLPl and the Cys-rich intestinal protein have only one LIM Gly-rich domain. Therefore, to determine whether single LIM Gly-rich domains of MLP may play a functional role in myogenesis, we stably expressed corresponding deletion constructs in C2 myoblasts. The structure of the LIMl and LIM2 constructs is shown in Figure 6A. These were devised to mimic the primary structure of DMLPl and the Cys-rich intestinal protein. As shown in Figure 6B, both LlMl and LIM2 promoted

representative cultures (n = 3: standard error, less than 10%) were normalized to one cell cycle of 18.5 hr. (D) Induction of the early myogenic marker muscle a-actinin in differentiating (2day) cultures are shown for CBMLP (a) and COASMLP (b). (E) Reducedsensitivityof C2-MLPcellstotheinhibitoryeffectofTGFpl on myogenic differentiation. Values are total contents of MHC-positive cells in 4day differentiating cultures expressed as percentage of the value in C2 cells in the absence of inhibitors. Values in parentheses are relative contents of weakly (+) stained cells. ND, not determined, but a value of about 10% is expected from similar determinations in the literature. Scale bar: 50 pm.

C hinTPA

_,

I

h-w

Figure 6. Like MLP, Single LIM Gly-Rich Deletion Mutants of MLP and the Nonmuscle LIM-Cnly Proteins CRP and Rhombotin-1 Promote Myogenic Differentiation in C2 Cells and Prevent TPA-Induced Inhibition of DNA Replication in 3T3 Fibroblasts (A) Schematic representation of MLP-based transfection constructs. The tag is a C-terminal epitope tag GLWMNIT (Arber et al., 1992). (6) Relative contents of MHC-expressing cells (all levels) in 3day differentiating cultures of stable C2 lines expressing the constructs as indicated. Abbreviations: rtn. mouse rhombotin-1 cDNA; C2, CPneo; AS-AChR, antisense rat acetylcholine receptor. (C) BrdU incorporation by stably transfected 3T3 cell lines in the absence and in the presence of 15 rig/ml TPA. Cells were plated at 20% confluency; TPA was added to the cultures 4 hr after plating and was removed 18 hr later. Transgenes: 1, LIMl; 2, CRP; 3, MLP; 4, LIM2; 5, insert-free vector; 6, GAP-43; 7, nontransfected 3T3 cells; 8, GAPXi(CC-to-AA). The GAP-43based control constructs are described elsewhere (Widmer and Caroni. 1993). (D) Matrix deposition in 24 hr cultures of 3T3-NE0 (insert-free vector) and 3T3MLP (A&P cDNA) cells. Double-labeling immunocytochemistry for tenascin (TN) and fibronectin (FN). Scale bar: 20 pm.

myogenic differentiation, although LIM2 was less effective than LIMl and both were less effective than MLP. These observations suggested that similar domains of nonmus-

cle LIM-only proteins may have a comparable activity when introduced into myogenic cells. We therefore performed analogous stable transfection experiments with the related (LIM Gly-rich)a protein CRP and with the (LIM)2 protein rhombotin-1. As shown in Figure 66, both CRP and rhombotin-1 promoted myogenic differentiation when expressed in C2 cells. Since nonmuscle LIM-only proteins can promote myogenie differentiation when expressed stably in C2 myoblasts, LIM-based regulatory mechanisms may also operate in cell types that express proteins related to MLP. CRP is expressed at low levels in quiescent fibroblasts and is an immediate-early gene when these cells are stimulated with serum. We therefore performed the reciprocal experiment and asked whether MLP, LlMl, or LIM2 may have effects comparable to those of CRP when stably

transfected into 3T3 fibroblasts. As shown in Figure 6C, CRP and the LIM-containing constructs all prevented TPAmediated inhibition of DNA replication. In addition, like those constitutively expressing CRP transgenes, MLPexpressing fibroblasts deposited a tenascin-rich and fibronectin-poor extracellular matrix onto the culture dish (Figure 6D). Therefore, LIM-only proteins and constructs have distinct effect8 when expressed in different cellular backgrounds. In addition, these findings suggest that LIMbased regulatory pathways may be widespread cellular control mechanisms. Discussion MLP is a novel and conserved LIM finger protein enriched in vertebrate striated muscle. In developing muscle and in myogenic cell lines, it is not found in proliferating myoblasts, and its expression and accumulation in the nucleus coincide with the formation and growth of myotubes. The

r2\scle

LIM Protein Promotes

Myogenic

Differentiation

embryonic expression of a Drosophila homolog of MLP, DMLP 7, is highly enriched in body wall and visceral muscles. DMLPl transcripts are first detectable in the myogenie mesoderm at the beginning of myogenic differentiation. This expression pattern is reminiscent of that of its vertebrate homolog MLP. Stable expression of an antisense MLP construct in C2 cells suppresses MLP expression and prevents myogenic differentiation in these cells. On the other hand, forced overproduction of MLP in C2 myoblasts potentiates myogenic differentiation upon transfer to differentiation-promoting culture medium. Potentiation of myogenic differentiation is also observed when single LIM Gly-rich domains of MLP or nonmuscle LIM-only proteins are stably expressed in C2cells, indicating that the LIM domains are relevant functional groups involved in the myogenic activity of MLP. Myogenically active LIM proteins induce a distinct and characteristic set of responses when expressed in 3T3 fibroblasts, suggesting the existence of widespread LIM finger-based regulatory mechanisms. Role of MLP in Myogenesis The stable transfection experiments of this study indicate that MLP plays an essential role in promoting the myogenic differentiation of C2 cells. In addition, the expression pattern of MLP and that of its Drosophila homolog DMLPl strongly support the notion that this protein plays a role in striated muscle development. What type of myogenic processes may be affected by MLP? The results of the differentiation experiments with transfected C2 cells indicate that MLP affects central regulatory aspects of myogenie differentiation. Thus, myogenin and clone 4 induction were not, and muscle a-actinin induction was only partially affected by the levels of MLP in C2 myoblasts responding to low serum conditions. These early myogenie reactions probably do not involve muscle-specific expression mechanisms, and up-regulation of myogenin in low serum is only weakly affected byTGF91. In contrast, withdrawal of differentiating myogenic cells from the cell cycle was sensitive to MLP and TGFPl , and CP-MLP cells were less sensitive to inhibition by TGFf31. Significantly, in spite of a normal induction of myogenin expression, this key event in myogenic differentiation was greatly reduced in the absence of MLP. After exit from the cell cycle, myogenie differentiation is no longer affected by TGFPl , while it remains sensitive to MLP, as indicated by the consistent potentiation of myotube growth in CP-MLP cells. Therefore, MLP appears to affect a regulatory process controlling muscle-specific gene expression. One possibility is that MLP may be acofactor involved in the muscle-specific activities of transcriptional activators like MDFs and MEF2. However, if this is the case, it may not involve a direct participation of MLP in the formation of transcription activation complexes binding to muscle-specific promoters since we could not detect an effect of MLP in gel retardation assays with myogenin and MEF1 DNA and since cotransfection of MLP with myogenin in C2 cells did not potentiate transcription from a muscle-specific MCK-CAT enhancer-promoter construct (data not shown). A potentiating effect by MLP in these transient transfection assays

may, however, have been masked by the adverse effects that very high levels of MLP have on myogenic differentiation in transient transfection experiments (see Results). In summary, our data indicate that myogenic differentiation is dependent on the presence of MLP after the induction of myogenin. Considering the broad stimulatory effects of MLP on myogenesis, our findings are consistent with the interpretation that MLP may affect a central regulatory pathway controlling muscle-specific gene expression. Elucidating the molecular mechanisms involved in its activity may have important implications to our understanding of skeletal and heart muscle growth under normal and pathological conditions. Structural Properties of MLP and Possible Roles of Its LIM Fingers MLP consists of two LIM Gly-rich domains that are separated by a spacer region of 35 amino acids. The two LIM Gly-rich domains have similar structures, including striking similarities between the first and second zinc fingers of each LIM structure. Accordingly, LlMl and LIM2 consist of the repeated zinc finger structures A0 and A’S’, respectively. The experiments with the single domain deletion mutants support the hypothesis that the LIM Gly-rich domains play a central role in the activity of MLP and that they may function independently. DMLPl consists of only one LIM Gly-rich domain, and our results suggest that it may play a similar essential role in myogenic differentiation in Drosophila. The forced expression experiments with the nonmuscle LIM-only proteins further support the interpretation that it is indeed the LIM finger motifs that mediate the activity of MLP. Surprisingly, the results show that quite different LIM fingers, as those of rhombotin-1 and MLP or CRP, are active in our assay. Specificity may, however, exist under more physiological conditions. In addition, the fact that in transfection experiments MLP and CRP accumulate in the nucleus and along actincontaining filaments in the cytosol, whereas LIMl, LIM2, and the (LIM)2 protein rhombotin-1 accumulate in the nucleus but do not not associate with actin filaments (S. A. and P. C., unpublished data), suggests that LIM protein activity may be controlled through regulated subcellular localization. The observation that MLP, CRP, LIMl, and LIM2 have common and characteristic activities when stably transfected in either myoblasts or fibroblasts suggests that LIM motif-based interactions may be a widespread mechanism regulating cell differentiation events. Two studies have begun to address the function of LIM finger motifs directly. The first study demonstrated that the LIM fingers of the homeodomain protein Lmx-1 allow it to form a minienhancer required for efficient and cell type-specific expression of the insulin gene (German et al., 1992). To achieve this, the LIM fingers of Lmx-1 apparently interact with a bHLH protein that binds to an adjacent site on the insulin promoter. This observation may be particularly relevant to this study, as MDFs are bHLH proteins. In the second study, the LIM motifs of Isl-l were shown specifically to prevent binding of this protein to a homeodomainbinding site (Sanchez-Garcia et al., 1993). This inhibition was abolished upon either proteolytic removal of the LIM

Cell 230

finger domains or chelation of zinc. In addition, construction of a chimeric LIM finger-Ultrabithorax homeodomain protein abolished the specific DNA binding activity of Ultrabithorax, indicating that LIM-mediated inhibition can be transferred to an heterologous homeodomain protein (Sanchez-Garcia et al., 1993). These studies and our results strongly implicate LIM fingers in key regulatory interactions involved in cell-specific gene expression. With respect to LIM-only proteins, they may regulate similar molecular processes controlling the expression of differentiation-associated genes in different cellular backgrounds. Induction of MLP expression at the onset of myogenie differentiation would provide this positive regulatory element for striated muscle formation. Experimental Procedures Reagents, Ceil Lines, and Antibodies Mouse myogenic C2C12 cells, human RD cells, Swiss mouse embryo fibroblasts (3T3), and lOT1/2 cells were from the American Type Cell CultureCollection. Theantibodyagainst theC-terminaipeptideof MLP (GGLTHQVEKKE) was produced by coupling the peptide to keyhole limpet hemocyanine (Sigma Chemical Company) via an N-terminal Cys. Monocional antibodies were obtained from the following sources: the muscle creatine kinase, muscle a-actinin, and myosin heavy chain were from Sigma Chemical Company, myomesin was from T. Wallimann (Swiss Federal institute of Technology, Zurich, Switzerland), tenascin was from Ft. Chiquet-Ehrisman (Friedrich Miescher Institute, Easel, Switzerland), and BrdU was from Becton Dickinson immunocytometry Systems. TPA was from Sigma and was used at a concentration of 15 nglml. TGF61 and bFGF were from Boehringer Mannheim. The mouse muscle creatine kinase probe was a gift of J. C. Perriard (Swiss Federal Institute of Technology, Zurich, Switzerland) (Jaynes et al., 19S6). The myogenin probe was a gift of A. Buonanno (National Institutes of Health, Bethesda, MD). The MCK-CAT plasmid was a gift of E. N. Olson (M. D. Anderson Cancer Center, Houston, TX). The coding region of human CRP (Wang et al., 1992) was cloned by PCR from a human fibroblast cDNA library (Stratagene).

lsoiation of Clones A subtracted cDNA library enriched in genes induced in denervated skeletal muscle was constructed according to a published procedure (Rubenstein et al., 1991). Two directional plasmid cDNA libraries from adult rat diaphragm (7 days hemidenervated from the synaptic region and innervated from the nonsynaptic region of the diaphragm) were constructed in Bluescript SK(+)derivatives(thegiftof D. Denney, Stanford University, Stanford, CA). Individual clones enriched in denervated diaphragm were selected. Two independent Drosophila DMLPl cDNA clones were isolated from a 12-24 hr embryonic cDNA library (provided by T. Kornberg, University of California, San Francisco) using a random-primed probe from the full-length rat MLP clone. Wholemount in situ hybridizations were carried out according to the protocol of Tautz and Pfeifle (1969), using random-primed digoxigenin-labeled probes (Boehringer Mannheim) from the entire DMLPl cDNA.

Blot Hybrtdlzation Procedures For Northern blots, equal amounts of total RNA were loaded on a formaldehyde gel, as verified by staining the blot with methytene blue. High stringency hybridization (66OC in 50% formamide. 5x SSC, 0.1% SDS, 0.1% N-lauroyisarcosine, 2.5% blocking reagent [Boehringer Mannheim], 100 pg/ml poiy[A]) with digoxigenin-labeled riboprobes and detection with AMPPD were performed as described by the manufacturer (Boehringer Mannheim). For genomic Southern analysis, hybridization was performed at 45OC overnight with a =Plabeled random-primed probe from the full-length rat MLP clone in 50% formamide-containing hybridization solution, and washes were twice for 40 min at 45OC and once for 40 min at 50°C in 1 x SSC, 1% SDS.

Ceil Culture, Trsnsfections, Ylcroinjactions, and tmmunocytochemistry The ceil lines 3T3, AD, and lOT112 were cultured in DMEM supplemented with 10% FCS. The cell line C2C12 was cultured either in DMEM, 10% FCS, 10% HS (GIBCO) (growth medium) or in DMEM, 2% FCS (differentiation medium). El 1 chick myotubes were prepared according to the protocol of Fischbach (1972). with slight modifications. For transfections, a CMV promoter-based eukaryotfc expression vector (pcDNA3; Invitrogen) containing a neomycin resistance gene for selection of stable clones was used. The tag sequence GLWMNIT was added to the C-terminus of MLP by two sequential PC&. Single LIM finger, CRP tag, and Rtn tag constructs were produced by an analogous procedure and verified by sequencing. Ceils were transfected using lipofectamine reagent (GIBCO). Following selection for 2-4 weeks, stable clones were pooled and analyzed for differentiation to myotubes, as described in Figure 4A. For most experiments described in this study, we analyzed several independent pools of clones. For immunocytochemistry, cells were rinsed with prewarmed PBS, permeabilized with PBS, 0.1% saponin (Sigma) for 15 s, and subsequently fixed in PBS, 3.7% formaldehyde for 20 min. Fixative was then removed, and cells were further permeabiiized with PBS, 0.1% Triton X-l 00 (Fluka) for 5 min and incubated in blocking solution (PBS, 5% BSA; Sigma) for IO min. Subsequent incubations with primary and secondary antibodies were performed in blocking solution. BrdU (and DAPI) labeling and detection were performed as recommended by the manufacturer (Boehringer Mannheim). For visualization of matrix deposition, 24 hr cultures were incubated for 3 min at room temperature in PBS, 0.5% Triton X-100,20 m M NH,OH (Bashkin et al., 1969). Subsequent antibody incubations were carried out in PBS, 5% BSA. Cryostat sections of unfixed adult rat tissue were fixed for 20 min at room temperature in PBS, 3.7% formaldehyde and then processed for immunocytochemistry in the absence of detergent. Levels of MHC production were defined as follows. In ceils producing high levels of MHC (+++), MHC-containing single fiber structures were not detectable at a 500-fold magnification, and characteristic MHCcontaining aggregate structures were detected by immunocytochemistry (for one such example, see Figure 4Ca). Weakly stained cells (+) had MHC signal levels lower than a set value. as determined with a videocamera.

Acknowledgments We are grateful to E. A. Nigg, M. Ruegg, G. Thomas, D. Monard, L. Aigner, and J.-M. Zingg for critical comments on the manuscript. The Drosophila work was carried out in W. Gehring’s laboratory at the Biocenter of Basel, and we thank him for his generosity. We thank M. Bethke, F. Botteri, P. Kuery, D. Denney (Stanford University), J. Rubenstein (University of California, San Francisco), A. Wiederkehr, U. Kioter, U. Walidorf, H.-R. Brenner, and S. Rotzler for assistance with some of the experiments. We are very grateful to C. Schneider for substantial help with the experiments. We are especially grateful to the numerous colleagues who generously provided us with precious reagents; their names can be found in Experimental Procedures. S. A. gratefully acknowledges the financial support of the Swiss Foundation for Research on Muscle Diseases. 0. H. thanks the Swiss National Foundation for Scientific Research and the Kantons of Base1 for financial support. Received January

17, 1994; revised August 16, 1994

References Arber, S., Krause, K. H., andcaroni, P. (1992). sCyclophilin is retained intraceiiuiarly via a unique CDDH-terminal sequence and coiocaiizes with the calcium storage protein calreticulin. J. Ceil Biol. 7 76, 113 125. Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn, M. C., Folkman, J., and Vlodavsky, I. (1969). Basic fibrobiast growth factor binds to subendothelial extracellular matrixand is released by heparitinase and heparin-like molecules. Biochemistry 28 , 1737-1743. Benezra, R., Davis, Ft. L., Lockshon, D., Turner, D. L.. and Weintraub,

Muscle LIM Protein Promotes 231

Myogenic

Differentiation

H. (1990). The protein Id: a negative regulator of helix-loop-helix binding proteins. Cell 67, 49-59.

DNA

Bengal, E., Ransone, L., Scharfmann, R., Dwarki, V. J., Tapscott, S. J., Weintraub, H., and Verma, I. (1992). Functional antagonism between cJun and MyoD proteins: a direct physical association. Cell 68, 507519. Boehm, T., Greenberg, J. M., Buluwela, L., Lavenir, I., Forster, A., and Fiabbitts, T. H. (1990). An unusual structure of a putative T cell oncogene which allows production of similar proteins from distinct mRNAs. EMBO J. 9,857-668.

Sadler, I., Crawford, A. W., Michelsen, J. W., and Beckerle, M. C. (1992). Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. J. Cell Biol. 119, 1573-1587. Sanchez-Garcia, I., Osada, H., Forster, A., and Rabbit&, T. H. (1993). The cysteine-rich LIM domains inhibit DNA binding by the associated homeodomain in 1~1-1.EMBO J. 72, 4243-4250. Sassoon, D. A. (1993). Myogenic regulatory factors: dissecting their role and regulation during vertebrate embryogenesis. Dev. Biol. 758, 11-23.

Campos-Ortega, J. A., and Hartenstein, V., eds. (1985). The Embryonic Development of Drosophila melanogaster (Berlin: SpringerVerlag).

Schwarz, J. J., Chakraborty, T., Martin, J., Zhou, J., and Olson, E. N. (1992). The basic region of myogenin cooperateswith two transcription activation domains to induce muscle-specific transcription. Mol. Cell. Biol. 12, 266-275.

Crawford, A. W., Pino, J. D., and Beckerle, M. C. (1994). Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton. J. Cell Biol. 724, 117-127.

Tautz, D.. and Pfeifle, C. (1989). A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophile reveals translational control of the segmentation gene hunchback. Chromosoma 98, 61-65.

Edmondson, D. G., and Olson, E. N. (1993). Helix-loop-helix proteins as regulatorsof muscle-specific transcription. J. Biol. Chem. 288,755758.

Wang, X., Lee, G., Liebhaber, S. A., and Cooke, N. E. (1992). Human cysteine-rich protein: a member of the LIM/double-finger family displaying coordinate serum induction with c-myc. J. Biol. Chem. 287, 9176-9164.

Fischbach, G. D. (1972). Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev. Biol. 28, 407429. Florini, J. R., Ewton. D. Z., and Magri, K. A. (1991). Hormones, growth factors, and myogenic differentiation. Annu. Rev. Physiol. 53, 201216. German, M. S., Wang, J., Chadwick, R. B., and Rutter, W. J. (1992). Synergistic activation of the insulin gene by a LIM-homeodomain protein and a basic helix-loop-helix protein: building of a functional insulin minienhancer complex. Genes Dev. 8, 2165-2176. Gu, W., Schneider, J. W., Condorelli. G., Kaushal, S., Mahdavi, V., and NadaCGinard, 6. (1993). Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation. Cell 72, 309-324. Jaynes, J. B., Chamberlain, J. S., Buskin, J. N., Johnson, J. E., and Hauschka, S. D. (1966). Transcriptional regulation of muscle creatine kinase gene and regulated expression in transfected mouse myoblasts. Mol. Cell. Biol. 8, 2855-2664. Lassar, A. B., and Weintraub, H. (1992). The myogenic helix-loop-helix family: regulatorsof skeletal muscle determination and differentiation. In Transcriptional Regulation (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), pp. 1037-1061. Li, L., Chambard, J. C., Karin, M., and Olson, E. N. (1992). Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus Of Jun can mediate repression. Genes Dev. 8, 676-669. Lyons, G. E., and Buckingham, M. E. (1992). Developmental regulation of myogenesis in the mouse. Semin. Dev. Biol. 3, 243-253. Martin, J. F., Li, L., and Olson, E. N. (1992). Repression of myogenin function by TGF51 is targeted at the basic helix-loop-helix motif and is independent of E2A products. J. Biol. Chem. 267, 10956-10960. Michelsen, J. W., Schmeichel, K. L., Beckerle, M. C., and Winge, D. R. (1993). The LIM motif defines a specific zinc-binding protein domain. Proc. Natl. Acad. Sci. USA 90, 4404-4406. Mizuno, K., Okano, I., Ohashi, K., Nunoue, K., Kuma, K.-l., Miyata, T., and Nakamura, T. (1994). Identification of a human cDNA encoding a novel protein kinase with two repeats of the LlMldouble finger zinc motif. Oncogene 9, 1605-1612. Nguyen, H. T., Medford, R. M., and NadaCGinard, B. (1983). Reversibility of muscle differentiation in the absence of commitment: analysis of a myogenic cell line temperature-sensitive for commitment. Cell 34, 261-293. Olson, E. N. (1992). Interplay between proliferation and differentiation within the myogenic lineage. Dev. Biol. 154, 261-272. Rubenstein, J. L. R., Brice, A. E., Ciaranello, R. D., Denney, D., Porteus, M. H., and Usdin, T. B. (1991). Subtractive hybridization system using single-stranded phagemids with directional inserts. Nucl. Acids Res. 18,4633-4842.

Weinlraub, H., Dwarki, V. S., Verma, I., Davis, R., Hollenberg, S., Snider, L., Lassar, A., and Tapscott, S. J. (1991). Muscle-specific transcriptional activation by MyoD. Genes Dev. 5, 1377-1366. Widmer, F., and Caroni, P. (1993). Phosphorylation-site mutagenesis of the growth associated protein GAP-43 modulates its effects on cell spreading and morphology. J. Cell Biol. 120, 503-512. GenBank

Accession

Numbers

The accession numbers for the rat MLP and DMLP 1 sequences ported in this paper are X61 193 and X81 192, respectively.

re-