Overexpression of Kelch domain containing-2 (mKlhdc2) inhibits differentiation and directed migration of C2C12 myoblasts

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Overexpression of Kelch domain containing-2 (mKlhdc2) inhibits differentiation and directed migration of C2C12 myoblasts Petra Neuhaus a,⁎, Benjamin Jaschinsky a , Sebastian Schneider a , Herbert Neuhaus a , Annelies Wolter a , Henning Ebelt b , Thomas Braun a,c a

Institute of Physiological Chemistry, MLU Halle-Wittenberg, Hollystr. 1, D-06114 Halle, Germany Institute of Physiological Chemistry and Department of Medicine III, MLU Halle-Wittenberg, Hollystr. 1, D-06114 Halle, Germany c Max-Planck Institute for Heart and Lung Research, W. G. Kerckhoff-Institute, Department of Cardiac Development and Remodeling, Parkstr. 1, D-61231 Bad Nauheim, Germany b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Targeted migration of muscle precursor cells to the anlagen of limb muscles is a complex

Received 14 December 2005

process, which is only partially understood. We have used Lbx1 mutant mice, which are

Revised version received

unable to establish correct migration paths of muscle precursor cells into the limbs to

12 May 2006

identify new genes involved in the accurate placement of myogenic cells in developing

Accepted 5 June 2006

muscles. We found that mKlhdc2 (Kelch domain containing-2), a novel member of the family

Available online 15 June 2006

of Kelch domain containing proteins, is significantly downregulated in Lbx1 homozygous mutant embryos. Functional characterization of mKlhdc2 by targeted overexpression in

Keywords:

10T1/2 fibroblasts and C2C12 muscle cells rendered these cells unable to respond to

Lbx1

chemoattractants such as HGF. Furthermore, C2C12 myoblasts overexpressing mKlhdc2

mKlhdc2

display altered cellular morphology and are unable to differentiate into mature myotubes.

Myoblast differentiation

Our results suggest that a tightly controlled expression of mKlhdc2 is essential for a faithful

Myoblast migration

execution of the myogenic differentiation and migration program.

Cytoskeleton

© 2006 Elsevier Inc. All rights reserved.

Differentiation

Introduction All skeletal muscles of the vertebrate body and some of the head muscles are derived from the somites. Somites are transient metameric structures that are laid down in a rostral to caudal direction on each side of the neural tube and notochord during embryogenesis. During further development, the somites will give rise to the sclerotome and the

dermomyotome [1,2]. Due to inductive and repressive instructions from surrounding tissues, the somite acquires a dorsal–ventral, anterior–posterior and medial–lateral polarity. Cells in the dorsal portion of the somite form the dermomyotome, which will develop into the dermis of the skin and the skeletal muscles of the trunk. The dermomyotome is further separated into a medial and a lateral population of cells, which will form epaxial (deep muscles of the back) and

⁎ Corresponding author. Current address: University Leipzig, Coordination center for clinical trials, Härtelstr. 16-18, D-04107 Leipzig, Germany. Fax: +49 0341 97 16109. E-mail address: [email protected] (P. Neuhaus). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.06.006

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hypaxial muscles (appendicular musculature, abdominal muscles, diaphragm, hypoglossal chord) respectively [1,3]. Formation of hypaxial muscle precursor cells (mpcs) at the ventro-lateral dermomyotomal lip depends on the expression of Pax3 (paired box 3) [4], which is also required for the correct expression of c-met, a receptor tyrosine kinase that in turn is responsible for the epithelial to mesenchymal transition leading to delamination of hypaxial mpcs from the dermomyotomal epithelium [3,5–9]. During their migration, hypaxial mpcs are kept in a proliferating and undifferentiated state and switch to the differentiation program only after arrival at their target destination (for reviews about muscle differentiation, see [10]). Since both Lbx1 and Pax3 are continuously expressed in mpcs [5,11,12] and strongly induce proliferation [13], it seems likely that these genes might maintain the migratory abilities of mpcs by delaying the onset of myogenic differentiation. Besides a tightly controlled expression pattern of transcription and differentiation factors in myogenesis, it has been assumed that re-organization of the cytoskeleton, formation of pseudopodia and other events, which are critical for the migration of other cell types, might also play a decisive role for the movement of mpcs [14–16], although so far very little is known about the “hardware” that enables migration of mpcs. Targeted mutation of Lbx1 leads to perinatal death and to drastically reduced appendicular muscle formation in homozygous mutant animals. Skeletal muscle is completely absent in the hind limbs of homozygous mutant animals, while the fore limbs are devoid of all muscle except proximo-ventral flexor muscles [17–19]. However, the tongue and diaphragm, which also belong to the hypaxial muscles, are not affected by the mutation, demonstrating that the pathfinding of different muscle precursor cells depends on more than one factor. Hypaxial muscle precursor cells in Lbx1 mutant mice delaminate from the dermomyotome and initiate migration but are unable to find their correct way and do not enter the limb bud for muscle formation. Instead of invading the limb buds, Lbx1 mutant cells accumulate at ectopic positions in the lateral plate mesoderm and later seem to adopt new cell fates according to their ectopic position [19]. Based on the analysis of Lbx1 mutant mice, it seems clear that Lbx1 is indispensable for the correct pathfinding of a large subset of limb muscle precursor cells and that loss of Lbx1 makes mpcs either “blind” for guidance to the correct target area or disables their long-range migration apparatus. Since the remaining muscle fibers in the forelimb of Lbx1 mutant mice do not mature entirely, it also seems feasible that Lbx1 is involved in the regulation of myogenic differentiation. In addition, ectopic expression of Lbx1 in chicken embryos activates myogenesis by an enlargement of the mpc population, further supporting the idea of a role for Lbx1 in the regulation of mpc proliferation versus differentiation [13]. Here, we have used the Lbx1 mutant mouse line created in our laboratory to identify downstream targets of Lbx1 and to define its role for myogenic differentiation and migration more precisely. We identified mKlhdc2, a novel mouse gene, as an indirect target of Lbx1 and analyzed its

function in muscle cell development and migration by different in vitro assays. Directed expression of mKlhdc2 in a muscle precursor cell line (C2C12) inhibited myoblast proliferation as well as differentiation, while proliferation of 10T1/2 fibroblasts was enhanced. In addition, the ability of both 10T1/2 fibroblasts as well as C2C12 myoblasts to respond to chemotactic cues was efficiently blocked by mKlhdc2. We conclude that mKlhdc2 plays an essential role in muscle cell migration and differentiation, potentially by acting on the cytoskeleton remodeling of myoblasts.

Materials and methods Tissue culture, transfection and FACS analysis C2C12 cells and 10T1/2 cells were transfected using Lipofectamine (Clontech) according to the manufacturer's instructions. A full-length mKlhdc2 expression plasmid (pCMV-Sport6mKlhdc2) was obtained from the RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH) and co-transfected in combination with the hygromycin resistance gene under the control of the PGK promoter. After 8 days of selection, hygromycin-resistant colonies were picked and expanded. After expansion to 60 mm dishes, mRNA was isolated using the Micro Fast Track Kit (Invitrogen) according to the manufacturer's instructions, reverse transcribed and analyzed for mKlhdc2 expression by both semiquantitative and real time quantitative PCR (see below). In vitro differentiation of C2C12 cells was achieved by serum depletion of the cells (growth medium: 20% FCS in DMEM containing 4500 mg/l glucose, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate; differentiation medium: 5% horse serum in DMEM, containing 4500 mg/l glucose, 100 U/ml penicillin and 100 μg/ml streptomycin sulfate) for 7 days. For cell adhesion assays, identical cell numbers of each analyzed cell clone were plated on tissue culture dishes and incubated for 20 min at 37°C before they were washed carefully and incubated at 37°C for another hour. Adherent cells were counted after the second incubation period. Trypsinization assays were performed by counting cells in several different areas of 90% confluent tissue culture plates after trypsinization for 5 min at 37°C followed by a washing step. The remaining, still adherent cells were incubated for 3 h at 37°C before they were counted. Each experiment was performed in eight independent wells and evaluated statistically using the Student's t test. DNA content measurements and cell cycle analyses by FACS were performed using propidiumiodide staining according to Ebelt et al. [20].

Immunohistochemistry For DiI labeling, cells were grown to 90% confluency and treated with 1 μg/ml DiI (Cell-tracker CM-DiI, Molecular Probes) according to the manufacturer's instructions. Before using the cells in coculture experiments, cell labeling was controlled under a fluorescence microscope. Each assay was performed in duplicate.

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Phalloidin staining was performed with Phalloidin-FITC (Sigma) at a concentration of 50 μg/ml in PBS. Nuclear labeling was achieved using either 4′-6-diamidino-2-phenylindole (DAPI, Sigma) or Hoechst 33258 (Molecular Probes) according to the manufacturer's instructions. All antibodies used were diluted in 1% horse serum in PBT. For myosin heavy chain staining, a hybridoma derived monoclonal antibody (MF20, Developmental studies hybridoma bank) was used. Hic5 detection was performed using a Hic5 antibody from BD Biosciences (dilution: 1/250). Secondary antibodies employed were Alexa Fluor 594 and Alexa Fluor 488 (goat anti mouse, Molecular Probes, Eugene, OR, diluted 1/1000).

Migration assays Migration assays were set up in a 48-microwell chemotaxis chamber (Neuro Probe, Cabin John, MD) with a polycarbonate membrane (pore size 8 μm). The lower chamber was filled with growth medium alone or growth medium containing 10 ng/ml IGF or 15 ng/ml HGF respectively. The upper chamber was filled with 50 μl of a cell suspension containing 1.6 × 104 cells/ml

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growth medium. The cells were incubated at 37°C for 16 h in a humidified incubator with 10% CO2. After incubation, the filter was washed and fixed according to the manufacturer's instructions. Cells were stained using Hoechst 33258, and the cell number was determined after photographing at a Nikon Eclipse E600 fluorescence equipped microscope using an area analysis software (SCION). Each assay comprising six wells was performed in triplicate and evaluated statistically using the Student's t test.

Hybridization of Affymetrix DNA microarrays, RT-PCR and real time quantitative PCR mRNA was isolated from E10.5 embryos or confluent 60 mm tissue culture dishes using the Micro Fast Track Kit (Invitrogen) according to the manufacturer's instructions. Affymetrix DNA microarrays (GeneChip Mouse Expression set 430) were hybridized, processed and analyzed using standard procedures at the OHRI gene expression center in Toronto, Canada. Reverse transcription and quantitative real time PCR was performed as described previously [21]. The sequences of the oligonucleotides used for amplification

Fig. 1 – (I) Semiquantitative RT-PCR showing mKlhdc2 and LZIP expression in differentiated and undifferentiated C2C12 cells and in 10T1/2 cells. GAPDH-PCR (glycerinaldehyde-3-phosphate) is shown as a control for cDNA synthesis. WT: wildtype cells, 10T-A: 10T1/2mKlhdc2-A, 10T-E: 10T1/2mKlhdc2-E, C2-A: C2C12mKlhdc2-A, C2-H: C2C12mKlhdc2-H, undiff.: undifferentiated, grown in growth medium, diff.: differentiated, grown for 7 days in differentiation medium. (II) Graph showing the results of a q-RT-PCR analysis performed to determine the relative expression levels of mKlhdc2 during myogenesis in wildtype and mKlhdc2-overexpressing cells. The expression level was normalized to wildtype C2C12 cells at day zero of differentiation.

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Table 1 Clone name

C2C12mKlhdc2A C2C12mKlhdc2B C2C12mKlhdc2C C2C12mKlhdc2D C2C12mKlhdc2E C2C12mKlhdc2F C2C12mKlhdc2G C2C12mKlhdc2H C2C12mKlhdc2I C2C12mKlhdc2J C2C12mKlhdc2K C2C12mKlhdc2L C2C12mKlhdc2M C2C12eGFP-A C2C12eGFP-B C2C12eGFP-C C2C12eGFP-D C2C12eGFP-E C2C12eGFP-F C2C12eGFP-G

eGFP mKlhdc2 expression expression

Differentiation Elongation Myotube of the cell formation body

n.a.

X

X

n.d.

n.a.

X

X

n.d.

n.a.

X

n.d.

n.d.

n.a.

X

X

n.d.

n.a.

X

n.d.

n.d.

n.a.

X

(X)

n.d.

n.a.

X

n.d.

n.d.

n.a.

X

n.d.

n.d.

n.a.

X

n.d.

n.d.

n.a.

X

n.d.

n.d.

n.a.

X

X

n.a.

X

n.d.

Few, very short n.d.

n a.

X

X

n.d.

X

n.d.

X

X

X

n.d.

X

X

X

n.d.

X

X

n.d.

X

X (reduced number and diameter) X

X

n.d.

X

X

X

n.d.

X

X

X

n.d.

X

X

The table gives an overview on the cell lines established ectopically expressing mKlhdc2 or eGFP and their differentiation capacities as judged by histological analyses (n.a.: not applicable, n.d.: not detectable).

were: Klhdc2 fwd 5′-GCT AAC AAT CTG CTG GTT C-3′; Klhdc2 rev 5′-TTC TCC ATG CAA GTG GAC-3′ (amplification of a 250 bp Klhdc2 fragment); LZIP 2 5′-CGG AAC AGG AGA TGT CTA GGC-3′; LZIP 4 5′-TAC AGA CAG GAG TCC TCA GGC-3′ (amplification of a 817 bp LZIP fragment). The oligonucleotide sequences for detection of muscle regulatory factors were taken from [22].

Loss of function analysis using mKlhdc2-RNAi oligonucleotides C2C12 cells were transfected with three different mKlhdc2-RNAi oligonucleotides or the block-it fluorescent oligo™ as a control

using Lipofectamine2000™ (Clontech), according to the manufacturer's instructions. Transfection was performed on 60 mm dishes at 40% confluency of the cells. The sequences of the used RNAi molecules were as follows: mKlhdc2-Ex3RNAi: 5′-CAA ACA AGU UCU ACA UGC UTT-3′; mKlhdc2-Ex6RNAi: 5′-GAA UUC AAG UCA UCC AAG ATT′-3′ and mKlhdc2-Ex13RNAi: 5′UAA CAA CAC UUC UGG AUC ATT-3′. The oligos were used in a concentration of 500 pmol/well (siKlhdc2) and 100 pmol/well (block-it fluorescent oligo™), respectively. The transfection efficiency was checked by fluorescence microscopy and was close to 90%. To estimate the knock-down effect of the RNAi oligos under growth conditions in C2C12 cells, cells were harvested 24 h after transfection and analyzed for mKlhdc2 expression by q-RT-PCR. To study the effect of RNAi knockdown of mKlhdc2 on the differentiation of C2C12 cells, the cells were cultured under differentiation conditions and transfected every 24 h for 5 days, with one complete medium change after 48 h. Subsequently, the cells were analyzed morphologically for cell fusion indicating myogenic differentiation and by q-RT-PCR for mKlhdc2 expression.

Results Isolation of mKlhdc2 We isolated homozygous Lbx1 and wildtype embryos and dissected the tissue regions located adjacent to the medial lip of the dermomyotome (fore- and hind limb regions). These regions are known to contain normal and mutant mpcs based on Lbx1-LacZ staining of Lbx1 heterozygous and homozygous reference embryos [19]. These tissues were used for RNA extraction and subsequent Affymetrix DNA microarray analyses (GeneChip Mouse Expression set 430) to identify putative Lbx1 target genes. Among several genes that were either up- or downregulated in Lbx1 mutant animals, including structural myogenic genes that would be expected, we identified mKlhdc2, an EST clone belonging to the family of Kelch domain containing proteins. The downregulation of mKlhdc2 in Lbx1 mutants was verified using quantitative RT-PCR. Since in situ hybridization experiments on whole embryos as well as on sections using various mKlhdc2 probes failed to yield a locally restricted signal in embryos at E9.5 to E11.5 (data not shown), we performed a comprehensive expression analysis by RT-PCR. mKlhdc2 is expressed at all embryonic stages and in all adult organs analyzed (E10.5, E 13.5, E16.5, E18.5, heart, brain, liver, kidney and muscle—data not shown) including the critical time window for the migration of mpcs into the limb buds. It seems likely that the ubiquitous expression of mKlhdc2 masks a specific detection in mpcs.

mKlhdc2 induces expression of LZIP and inhibits differentiation of C2C12 muscle cells Since mKlhdc2 is downregulated in Lbx1 mutant embryos and is present at the right time and the right place to control important aspects of muscle development, we turned to a functional analysis to begin to decipher the role of mKlhdc2 in cellular migration and differentiation. We decided to overexpress mKlhdc2 in 10T1/2 cells, a mouse mesenchymal

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cell line, which contains moderate levels of mKlhdc2 (Fig. 1) and in the muscle cell line C2C12, which is an established model to study myogenesis in vitro [23–25] and which expresses mKlhdc2 only after initiation of differentiation (Fig. 1). 10T1/2 cells were chosen as a control fibroblast cell line used for migration and proliferation tests, while C2C12 cells additionally served as a model for muscle cell differentiation. After hygromycin selection, we obtained 6 resistant 10T1/2 cell clones and 13 C2C12 cell clones (Table 1), which showed different expression levels of the exogenously introduced CMV-driven mKlhdc2 expression construct as determined by semiquantitative RT-PCR (Fig. 1 and data not shown). As a control, we transfected C2C12 cells with an expression plasmid containing eGFP instead of mKlhdc2 and obtained seven stable cell lines which showed the same characteristics like the parental, wildtype C2C12 cells (Table 1 and data not shown). After mKlhdc2 transfection, 10T1/2 as well as undifferentiated C2C12 cells displayed a clear induction of the expression of the basic leucine zipper transcription factor LZIP (Fig. 1), which has been described to be a target of posttranslational regulation of mKlhdc2 in HOS cells [26]. Thus, our data suggest that mKhldc2 regulates LZIP not only posttranslationally but also transcriptionally. Induction of differentiation by serum depletion revealed a strong inhibition of myogenic differentiation after directed expression of mKlhdc2. While parental C2C12 cells and C2C12

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cells transfected with the eGFP plasmid readily differentiated into MF20 positive myotubes (Fig. 2 and data not shown), a completely different picture emerged in mKlhdc2-overexpressing clones. All 13 C2C12 clones, which overexpressed mKlhdc2, were devoid of mature myotubes. Only those clones, which were characterized by a comparatively low expression of mKlhdc2, were sometimes able to generate single immature myocytes. Some cell lines still showed an elongation and a parallel orientation of single cells, reflecting the initial step of myotube formation, while other cell lines did not show any sign of differentiation (Table 1, Fig. 2 and data not shown). We therefore selected two representative cell lines, C2C12mKlhdc2-A and C2C12mKlhdc2-H, which showed a slightly different differentiation behavior for further analysis. Careful examination of the differentiation potential of these two representative isolates by RT-PCR indicated that mKlhdc2-overexpressing cells initially expressed the myogenic determination factors Myf5 and MyoD similar to the parental cell line. Upon differentiation, wildtype C2C12 cells downregulated Myf5 and MyoD and upregulated Myogenin parallel to the initiation of myotube formation. In contrast, mKlhdc2-overexpressing clones failed to do so but maintained Myf5 expression (Fig. 2). Since some mKlhdc2-overexpressing clones seemed to show initial steps of myotube formation such as elongation and parallel orientation of myoblasts, but failed to fuse and did not form a mature syncytium, we wanted to know if the observed phenotype was due to a fusion deficit rather than a

Fig. 2 – (I) Histological and (II) RT-PCR analysis of the differentiation capacity of mKlhdc2-overexpressing cell lines in comparison to wildtype cells. Phalloidin staining of the actin cytoskeleton is shown in green, MF20 staining for myosin heavy chain in red and Hoechst nuclear staining in blue. While wildtype cells clearly show formation of MF20 positive myotubes (arrow in C), while mKlhdc2-overexpressing cells do not differentiate (F, I). C2C12Klhdc2A cells still show parallel orientation of the cells (E, F), which is not detectable in C2C12Klhdc2H cells (H, I), indicating a total differentiation block of the latter cell line. The loss of differentiation is also reflected by the maintained Myf5 expression in mKlhdc2-overexpressing cell lines C2C12Klhdc2A and C2C12Klhdc2H after stimulation of differentiation and the lack of Myogenin induction. Undif.: undifferentiated, Diff.: differentiated, WT: wildtype, A: C2C12Klhdc2A, H: C2C12Klhdc2H; scale bar: 250 μM.

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differentiation block. We therefore labeled mKlhdc2-overexpressing cells with DiI and cocultured them in different ratios together with wildtype C2C12 cells. Depending on the ratio of wildtype to mKlhdc2-overexpressing cells, the engineered cells were able to participate in the formation of myotubes. In principle, we found that the number of DiI-labeled cells in mature, MF20-expressing, myotubes increased when more wildtype C2C12 myoblasts were added to the culture (Fig. 3) indicating that mKlhdc2 overexpression did not prevent recruitment of engineered cells into C2C12 cell derived myotubes, but inhibited cell autonomous differentiation. For control purposes, we also labeled wildtype C2C12 cells with DiI and performed the same coculture experiment as with the mKlhdc2-overexpressing cells. Labeled wildtype cells readily fused with each other and with unlabeled cells, indicating that the labeling procedure did not affect the fusion capacity and/ or differentiation of the C2C12 cells (supplemental figure available in Appendix A).

Enhanced expression of mKlhdc2 blocks HGF-mediated targeted migration Cellular migration is an essential process in hypaxial muscle development since hypaxial mpcs migrate long distances to reach their target area during embryogenesis. The migratory ability of mpcs is probably determined by the action of different cytokines such as HGF/SF and IGF [7,27]. We analyzed the effect of ectopic mKlhdc2 expression on the migration of 10T1/2 and C2C12 cells using the Boyden chamber assay. Wildtype cells and two representative mKlhdc2-overexpressing clones of 10T1/2 and C2C12 cells (10T1/2Klhdc2-A, 10T1/2Klhdc2-E, C2C12Klhdc2-A and C2C12Klhdc2-H) were incubated in a Boyden

Fig. 3 – mKlhdc2-overexpressing cells are able to fuse with wildtype cells. DiI-labeled overexpressing cells were mixed with wildtype cells in a ratio of 5/6 or 1/6 (WT/C2C12mKlhdc2-A A and C, WT/C2C12mKlhdc2-H B and D). Depending on the number of wildtype cells, more DiI derived from overexpressing cells was present in MF20 positive myotubes (arrows in A–D), while a lower number of wildtype cells restricted DiI labeling to undifferentiated, mKlhdc2-overexpressing cells (arrowheads in D). Blue: DAPI nuclear stain. Red: DiI, green: MF20/myosin heavy chain.

Fig. 4 – (I) In comparison to wildtype cells, cells overexpressing mKlhdc2 do not respond to HGF. (II) Neither wildtype nor mKlhdc2-overexpressing cells reacted to chemoattraction by IGF in our hands. The targeted migration of cells in a Boyden chamber assay as a multiple of migrating cells with chemoattractant (+) in comparison to without chemoattractant (−) is shown as relative migration normalized for each cell line independently based on cells migrating without chemoattraction (i.e. no chemoattractant added). HGF: hepatocyte growth factor, IGF: insulin like growth factor, WT: wildtype cells, 10T-A: 10T1/2mKlhdc2-A, 10T-E: 10T1/2mKlhdc2-E, C2-A: C2C12mKlhdc2-A, C2-H: C2C12mKlhdc2-H, significance was tested using the Student's t test, and significant differences are labeled by a star (p < 0.05).

chamber with IGF (insulin like growth factor), HGF (hepatocyte growth factor) and without a chemoattractant. The addition of IGF to the medium in the lower chamber had no significant effect on the migration of any of the cell lines used (Fig. 4). However, 10T1/2 and C2C12 wildtype cells showed a clear induction of migration upon supplementation of the medium with HGF/SF. In contrast to wildtype cells, mKlhdc2-overexpressing clones did not respond to the addition of HGF by enhanced migration (Fig. 4). These results clearly demonstrate that overexpression of mKlhdc2 led to a loss of HGF-mediated directed migration of 10T1/2 and C2C12 cells.

mKlhdc2 overexpression stimulates proliferation of 10T1/2 cells Since migrating mpcs are kept in a proliferative and undifferentiated state and hKlhdc2 as well as its target molecule LZIP have been associated with enhanced cell cycle progression, we wanted to explore whether overexpression of

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mKlhdc2 also affects cell cycle progression. We used FACS analyses to determine the proliferation rate of mKlhdc2overexpressing cells. Interestingly, overexpression of mKlhdc2 had opposite effects on the proliferation of 10T1/2 fibroblasts and C2C12 myoblasts. Directed expression of mKlhdc2 strongly stimulated proliferation of 10T1/2 cells as revealed by a clear reduction of the number of cells in G1 and an increase of cells in the S-phase (Fig. 5). The opposite effect was observed in C2C12 cells ectopically expressing mKlhdc2: the number of cells in the G1-phase was significantly higher accompanied by a decrease of cells in S-phase resulting in reduced cellular proliferation (Fig. 5).

Directed expression of mKlhdc2 leads to augmented stress fiber formation and cell adherence Careful morphological examination of C2C12 cells carrying the mKlhdc2 expression construct unveiled a “flattened” phenotype. This impression was further supported by the observation that ectopic mKlhdc2 expression led to a stronger adherence of the cells to tissue culture plates. mKlhdc2overexpressing C2C12 cells were harder to trypsinize and attached better to tissue culture plates than wildtype cells (Fig. 6). Phalloidin staining of actin filaments of parental and mKlhdc2-overexpressing cells indicated that the enhanced adhesion might be due to increased stress fiber formation and a potentially enhanced number of focal adhesion complexes. To corroborate this hypothesis, we used Hic5 antibody staining in combination with Phalloidin staining. Hic5 is a component of focal adhesion complexes, and an accumula-

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tion of Hic5 is indicative for an increase in focal adhesion complex formation. While stress fibers and focal adhesion complexes normally form at the periphery of a cell, mKlhdc2overexpressing cell lines showed an increased number of stress fibers and focal adhesion complexes which were evenly distributed over the complete cell body (Fig. 6). These findings further highlight the role of mKlhdc2 in actin remodeling, which is a hallmark of migrating cells.

Downregulation of mKlhdc2 by RNAi interference in C2C12 cells does not inhibit myogenic differentiation Since mKlhhdc2 is normally upregulated during myogenesis and the overexpression of mKlhdc2 in C2C12 myoblasts leads to a differentiation block, we wanted to know whether a reduction of mKlhdc2 expression affects the ability of C2C12 cells to differentiate. We transfected wildtype C2C12 cells with three different RNAi molecules targeted to the 3rd, 6th and 13th exon of mKlhdc2 respectively. Under growth conditions, all three oligos led to mKlhdc2 silencing (Fig. 7). This picture was lightly different under differentiation condition where only one of the three oligos clearly repressed mKlhdc2. Interestingly, the knock-down of mKlhdc2 cells did not affect the differentiation capacity of C2C12 cells. Morphological evaluation of the differentiated cells showed normal myotube formation in control cells (transformed with block-it fluorescent oligo or not at all) as well as RNAi-treated cells (Fig. 7).

Discussion

Fig. 5 – Cell cycle analysis of wildtype and mKlhdc2-overexpressing cell lines by FACS analysis. While C2C12 cells show reduced cellular proliferation as a consequence of mKlhdc2 overexpression, 10T1/2 cells show increased cell cycle progression after mKlhdc2 overexpression. Percentage of cells in S-, G1- or G2-phase after expression of mKlhdc2 was calculated using the MultiCycle software. *P < 0.05 (overexpressing cells compared to wildtype cells).

Based on an Affymetrix microarray assisted screening, we have identified mKlhdc2 as a potentially important component of the machinery that directs migration and differentiation of mpcs. Paradoxically, the upregulation of mKlhdc2 resulted in a block of myogenic differentiation and migration of myogenic cells in vitro. Since the reduced presence of mKlhdc2 in Lbx1 mutants correlated with the inability of Lbx1 mutant muscle precursor cells for targeted migration, one might have expected a stimulation of migration upon enhanced expression of mKlhdc2 and a block of migration after downregulation of mKlhdc2. Instead, we found an impairment of migration and muscle cell differentiation after overexpression of mKlhdc2. Since mKlhdc2 is known to affect the expression of various cellular targets by transcriptional and post-transcriptional mechanisms, we concluded that a faithful execution of the myogenic differentiation and migration program depends on a tightly controlled expression level of mKlhdc2. Another possible explanation for the observed downregulation of mKlhdc2 in Lbx1 mutant embryos is the loss of differentiated myogenic cells in the limbs of Lbx1 mutant mice rather than the absence of migratory muscle precursor cells. Since mKlhdc2 is strongly expressed in C2C12 myotubes and differentiated muscle cells, the absence of differentiated muscle cells (as in the limbs of Lbx1 mutant mice) will lead to a decreased concentration of mKlhdc2 in Lbx1 mutant limbs. Based on the expression profile of mKlhdc2, which shows an upregulation during muscle

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Fig. 6 – (I) wildtype cells show mostly peripheral focal adhesion complexes (indicated by Hic5) and stress fibers (Phalloidin staining). The inlay shows a lower magnification of the picture, with the rectangle indicating the area for the blow up. (a, d), C2C12mKlhdc2-A (b, e) and C2C12mKlhdc2-H (c, f) cells display increased stress fiber and focal adhesion formation across the cell body, which is most prominently seen in C2C12mKlhdc2-H cells (f). Red: Hic5 staining, green: actin/Phalloidin staining, blue: DAPI nuclear stain; scale bar: 250 μM. The enhanced number of stress fibers and focal adhesions led to a stronger adhesion competence (II) and lower trypsinization efficiency (III) of mKlhdc2-overexpressing C2C12 cells in comparison to wildtype cells. For II and III, significance was tested using the Student's t test, and significant differences are labeled by a star (p < 0.02).

differentiation (possibly to terminate migration), it seems likely that mKlhdc2 is an indirect and not a direct target of Lbx1. mKlhdc2 is a transcriptional cofactor, which so far has only been described as a mouse EST clone. It belongs to the family of Kelch domain containing proteins, which are known to be involved in such diverse biological functions as transcriptional activation or repression, actin remodeling or stabilization and cell adhesion (for a review, see [28]). The Kelch domain consists of four to seven β-sheets, which form a propellerlike structure that is essential for protein–protein interactions.

In the case of mKlhdc2, the propeller consists of six β-sheets and essentially covers the whole protein, with no other motives detectable by database searches. mKlhdc2 shows the highest homology to its human orthologue hKlhdc2 (also named HCLP1, [26]) and host cell factor 1 (HCF1). mKlhdc2 and hKlhdc2 are 79% identical on the nucleotide level and 96% identical on the protein level. So far, there are no data available that disclose the biological function of either mouse or human Klhdc2 in vivo. hKlhdc2 was originally cloned based on its similarity to HCF1 (host cell factor 1) [26]. In addition, it was isolated as a

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Fig. 7 – Quantification of the mKlhdc2 expression after gene silencing by siRNA in wildtype C2C12 cells: (I) under growth conditions, (II) under differentiation conditions. Shown is the relative mKlhdc2 expression level normalized to wildtype, non-transfected cells. Significant differences are labeled by a star (p < 0.08).

protein associated with hepatocellular tumors (HCA33, Gene bank AF244137), and thus it has been proposed to be involved in cellular proliferation and transformation events. Until now, Klhdc2 has not been connected to differentiation or migration events of any cell type. Interestingly, LZIP, a basic leucine zipper transcription factor and so far the only described cellular target of hKlhdc2, has been shown to improve targeted migration of HOS cells (human osteosarcoma cells) in vitro. Overexpression of LZIP in combination with CCR1 (chemokine (C-C motif) receptor 1, [29]) led to enhanced chemotactic migration of HOS cells in response to Lkn1 (Leukotaktin 1, [30]). It was assumed that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn-1-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1 [30]. Interestingly, hKlhdc2 has been shown to directly interfere with the DNA-binding activity of LZIP [26]. In addition, we have demonstrated that mKlhdc2 also leads to a transcriptional upregulation of LZIP. Hence, the reduced chemotactic response of cells expressing ectopic mKlhdc2 might be caused indirectly by

the induction of LZIP transcription and the activation and/or repression of cellular targets of LZIP rather than mKlhdc2 itself. In the current study, we found an increased number of stress fibers and focal adhesion complexes that were almost evenly distributed over the cell. Focal adhesions serve as transmembrane signaling centers due to the presence of proteins like FAK (focal adhesion kinase), paxillin, integrins and src [15] and are involved in actin remodeling and changes in the adherence of the cells. During myogenic differentiation, the activity of FAK is tightly controlled, reflecting the role of cellular adhesion for the regulation of myogenesis [31–33]. Based on the alterations in cellular morphology of mKlhdc2-overexpressing cells, it seems possible that mKlhdc2, potentially also via LZIP, might act in a more general way and affect actin remodeling. The significantly increased amount of stress fibers and focal adhesion complexes in mKlhdc2-overexpressing cells might be envisioned as a type of “cast”, potentially rendering cells unable to perform “normal” functions like cell division and cell remodeling, which are essential for cellular migration.

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In this context, it is interesting to note that the repression of mKlhdc2 induction (which normally occurs during differentiation of C2C12 cells) by an RNAi approach, did not affect the differentiation capacity of C2C12 cells. This is not surprising, since we assume that mKlhdc2 upregulation in differentiating myoblasts serves to alter the cytoskeleton and to stabilize forming myotubes against mechanical stress rather than to control myocyte differentiation directly. To obtain a more detailed insight into the biological function of mKlhdc2, it will be essential to assess the integrity of mKlhdc2 negative muscle fibers during myogenic development and in adult muscles. As mentioned above, hKlhdc2 was originally identified as a protein associated with hepatocellular tumors, which suggests no direct link to muscle development. However, it seems possible to draw a parallel between transformation of cells and differentiation/migration of mpcs. In both conditions, cells are essentially maintained in a proliferating and undifferentiated state and are kept mobile, preventing stable integration into an organized tissue. Like hKlhdc2, LZIP, which is induced by mKlhdc2, has also been shown to be involved in cellular transformation. In contrast to hKlhdc2, not gain, but a loss of LZIP function leads to oncogenic transformation [34]. It has been proposed that hKlhdc2 might act as a proto-oncogene, while LZIP suppresses tumor formation. According to this model, the oncogenic potential of hKlhdc2 results from the inhibition of LZIP [26,34]. These data suggest that mKlhdc2overexpressing cell lines might show enhanced cellular proliferation, though we detected enhanced cellular proliferation only in 10T1/2 cells, while C2C12 cells showed a decrease in cell cycle progression. The reason for this difference might be due to differences in the expression of Klhdc2 and LZIP in C2C12 and 10T1/2. 10T1/2 cells, in contrast to undifferentiated C2C12 cells, express robust levels of hKlhdc2 and LZIP, and additional expression of mKlhdc2 did not lead to a further increase of LZIP expression. We reason that the induction of LZIP expression, which might act as an antagonist to Klhdc2 with respect to cellular transformation, might counteract enhanced proliferation of C2C12 cells. According to the available data, both mKlhdc2 and LZIP potentially influence the expression, activity and localization of numerous molecules in the cell. It is clear that more subtle means and a dissection of the molecular functions of mKlhdc2 in vivo are required to obtain a more comprehensive picture of the role of mKlhdc2 for various biological processes including myogenic development.

Acknowledgments We thank Mechthild Hatzfeld (University of Halle-Wittenberg, Halle, Germany) for the Hic5 antibody, useful discussions and for help with the fluorescence microscope. This work was supported by the Deutsche Forschungsgemeinschaft, priority program “Cell migration”, the “Fonds der Chemischen Industrie” and the Wilhelm-Roux-Program for Research of the Martin-Luther-University.

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