Diacylglycerol kinase theta and zeta isoforms: regulation of activity, protein binding partners and physiological functions

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Los, Alrik Pieter

Citation

Los, A. P. (2007, January 25). Diacylglycerol kinase theta and zeta isoforms: regulation of

activity, protein binding partners and physiological functions. Retrieved from

https://hdl.handle.net/1887/9451

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the

Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/9451

Note: To cite this publication please use the final published version (if applicable).

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Diacylglycerol kinase- c

st im u lat es m u scle

d ifferent iat ion

Alrik P. Los

J oh n d e W id t

C a t e lijn e S t ort e le rs

F a b ia n P. V in ke

W im J . v a n B lit t e rsw ijk

N u llin D iv e c h a

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A b stract

W e p rev iously sh ow ed th at diacylglycerol kinase- c (

DG K

c ) ,

w h ich p h osp h orylates diacylglycerol (

DA G

) to p roduce

p h osp h atidic acid (

P A

) , b inds to and is activ ated b y th e

retinob lastoma p rotein ( p

R B

) and its family memb ers.

DA G

,

P A

and p

R B

are intimately imp licated in regulating

myogenesis and th is led us to determine if

DG K

c h as a

p h ysiological role in regulating muscle differentiation.

O v erex p ression of

DG K

c in

C 2 C 12

myob lasts enh anced

b oth muscle differentiation and th e ex p ression of muscle-

sp ecific genes, w h ich w as dep endent on th e activ ity of

DG K

c . Microarray analysis of gene ex p ression demonstrat-

ed th at many muscle-sp ecific genes w ere up regulated b y

th e ov erex p ression of

DG K

c . F urth ermore, many of th e

genes th at w ere up regulated w ere direct targets of th e

master myogenic transcrip tional regulator

M

yo

D

and w e

sh ow th at

DG K

c is ab le to stimulate transcrip tion from

th ree different

M

yo

D

dep endent p romoters. T o demon-

strate a p h ysiological function for

DG K

c in myogenesis,

w e sh ow th at mouse emb ryonic fib rob lasts (

ME F

s) deriv ed

from

DG K

c -null mice w ere unab le to differentiate into

muscle. O ur results sh ow th at

DG K

c h as an essential role

in muscle differentiation.

Diacylglycerol kinase-c stimulates muscle differentiation

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I ntroduction

Skeletal muscle differentiation is a highly ordered process that involves the sequen- tial activation of muscle-specific transcription factors. Muscle differen tiation is induced in culture by serum deprivation of proliferating myoblasts which ex it the cell cycle and fuse into multinucleated myotubes. T wo families of transcription factors play essential roles in muscle differentiation: the basis helix -loop-helix transcription factor family of myogenic regulating factors (MR Fs) and the mono- cyte enhancer factor 2 (MEF2 ) family of MADS-box transcription factors. Both families regulate one anothers ex pression and cooperate in the activation of many muscle-specific structural genes.

T he MR F family consists of MyoD, myogenin, Myf-5 and MR F4 that are each capa ble of inducing the complete muscle differentiation program when over- ex pressed in non-muscle cell types (Weintraub, 1 9 9 3 ) . All four myogenic factors heterodimer ise with E proteins thereby forming a functional complex that bind and activate E-box -containing promoters of muscle-specific genes (Lassar et al., 1 9 9 1 ) .MyoD and Myf-5 are ex pressed in proliferating myoblasts prior to the onset of muscle differentiation and regulate muscle lineage determination. Myogenin collaborates with MyoD and MEF2 to regulate ex pression of genes important for terminal differentiation, including myosin heavy chain (MH C) and muscle creatine kinase (MCK) .MR F4 is involved in both the early stages of differentiation and terminal differentiation (Berkes and T apscott, 2 0 0 5 ) .

Another key player in muscle differentiation is the retinoblastoma protein (pR B) . During differentiation, pR B levels are upregulated by MyoD that stimulates pR B promoter activity (Magenta et al., 2 0 0 3 ) . pR B is involved in two steps in the differentiation process: cell cycle ex it and ex pression of late differentiation genes. T he first key step in differentiation is induction of growth arrest in proliferating myoblasts. U pon differentiation, MyoD and Myf5 are involved in the induction of cyclin/CDK inhibitors, including p2 1WAF1 / Cip1, that downregulate cyclin/CDK complex es (K itz mann and Fernandez , 2 0 0 1 ) . Consequently, pR B becomes hypo- phosphorylated, which causes a cell cycle arrest. pR B-deficient myoblasts fail to withdraw from the cell cycle and do not differentiate properly (Novitch et al., 1 9 9 6 ) .Myogenin and p2 1WAF1 / Cip1 ex pression is not affected in pR B-deficient myoblasts, but late differentiation genes including MH C and MCK are not ex pressed.

Promoter studies of late differentiation genes revealed that pR B is required for MyoD and MEF2 -mediated promoter activation (Novitch et al., 1 9 9 6 ; Novitch et al., 1 9 9 9 ) . pR B is not required for maintaining differentiated myotubes in the postmitotic state, since myotubes in which pR B was ex cised do not re-enter S-phase (Camarda et al., 2 0 0 4 ; H uh et al., 2 0 0 4 ) . H owever, pR B is required for ex pression of latephase muscle-specific genes (Camarda et al., 2 0 0 4 ) .

We previously showed that diacylglycerol kinase-c (DG K c) , which phosphor- ylates the lipid second messenger diacylglycerol (DAG ) yielding phosphatidic acid (PA) , binds to and is activated by pR B and its family members p1 0 7 and p1 3 0 (Los et al., 2 0 0 6 ) . PK C is a physiological downstream target for DAG , whereas mT O R is activated by PA. Both PK C and mT O R are intimately involved in the regulation of

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myogenesis (Li et al., 1992; Liu et al., 1998; Hardy et al., 1993; Ohanna et al., 2005).

Therefore, we sought to determine if DGKc may play a role in muscle differentiation. We show here that C2C12 myoblasts overexpressing wild-type DGKc, but not kinase-inactive DGKc, stimulates muscle differentiation. Overexpression of DGKc enhances the expression of muscle-specific genes as revealed by mRNA microarray analysis. To demonstrate that DGKc expression is physiologically required for muscle differentiation we show that mouse embryonic fibroblasts isolated from DGKc-null mice do not differentiate into myoblasts in response to MyoD upregulation. Our results suggest that either the removal of DAG and/or the production of PA regulated by the activity of DGKc is important in myogenesis.

Materials and methods

Tr a n s d u c t i o n a n d d i f f e r e n t i a t i o n o f c e l l s

C2C12 cells were transduced with retroviral supernatants harvested from pBabe- puro-transfected ecotropic phoenix cells, as described (Los et al., 2006). After 24 hours, cells were selected with 2 +g/ml puromycin and were used when they stably expressed DGKc.C2C12 cells were seeded (100.000 per 6-well or 200.000 cells per 6 cm dish) and after 24 hours induced to differentiate in DMEM containing 2% horse serum. This so-called differentiation medium was refreshed after 3 days.

At different time-points of differentiation, cells were washed with cold PBS and snap-frozen.

DGKc^-/-- or wild-type mouse embryonic fibroblasts (MEFs) (kindly provided by M. Topham) were transduced with retroviral supernatants derived from MyoD- or empty vector-transfected Phoenix packaging cells as described in (Los et al., 2006).

Stable cell lines were seeded into 6-well plates (100.000 cells/well) and, after 24 hours, cells were stimulated to differentiate in differentiation medium containing 10 +g/ml insulin. At different time-points of differentiation, cells were washed twice with cold PBS and snap-frozen.

C e l l l y s i s a n d We s t e r n b l o t t i n g

C2C12 cells were lysed in RI PA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 158 mM NaCl, 10 mM Tris pH 7.2, 1 mM EGTA, complete protease inhibitor cocktail (Roche) and 30 +g lysate protein was separated using 4-15% criterion precast gels (Bio-Rad). Proteins were transferred to nitrocellulose and stained using an anti- DGKc polyclonal antibody (Bunting et al., 1996), anti-MyoD M-318 (Santa Cruz), anti-myogenin F5D or anti-myosin MF20 (Developmental Studies Hybridoma Bank).

Blots were stained with secondary antibodies (DAKO) and visualised using ECL.

R e a l - t i m e P C R

RNA was isolated from C2C12 cells at different time-points of differentiation using the RNeasy mini kit (Q iagen) according to manufacturer’s instructions. cDNA was synthesised using Superscript first-strand synthesis system (I nvitrogen) and was

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quantified by real-time PCR. Samples were run on an ABI Prism 7700 real-time PCR machine (Applied Biosystems) using SY BRgreen PCR master mix according to the manufacturer’s instructions. Reactions contained 300 nM of each primer.

The cycling parameters of the reactions were: 50°C for 2 min, 95°C for 10 min, and 50 cycles of 95°C for 15 s and 60°C for 1 min. To analyse melting curves, samples were ramped from 60°C to 95°C over a period of 19 min. Samples were analysed using the comparative C_T method in which samples were normalised to GAPDH expression. The fold-difference in expression was related to undifferentiated vector controls and standard deviations were used to calculate the range. Statistics were performed using the double delta model (DDM) (Hoogendam et al., 2006).

R e p o r t e r a s s a y s

C2C12 cells stably expressing DGKc or vector were seeded in 6-well plates (50.000 cells) and after 24 hours they were co-transfected with reporter constructs (kindly provided by V. Sartorelli) and CMV-renilla using Fugene. Transfection efficiency was checked in different wells by GFP-H2B transfection. 14-18 hours after transfection cells were washed ones and maintained in differentiation medium for 48 hours. Cells were washed with cold PBS and snap-frozen. For luciferase assays cells were lysed in 250 +l passive lysis buffer and 20 +l was used to measure luciferase activity, as described (Los et al., 2006).

M i c r o a r r a y s

Detailed information about the microarray system is shown on http://microarrays.

nki.nl. Total RNA was isolated from C2C12 cells at 5 days of differentiation and amplified using the superscript RNA amplification system (Invitrogen). Amplified RNA was labeled with ULS-Cy5 or ULS-Cy3 using the ULS aRNA labeling kit (Kreatech Biotechnology). Blocking reagents (poly-dA (Pharmacia), human COT- 1DNA (Invitrogen), and tRNA (Roche)) was added to the amplified RNA and this mixture was subsequently diluted in 2 x F-hybridization buffer (final concentration:

25% formamide, 5× SSC, and 0.1% SDS).RNA was hybridised to oligo micro arrays and subsequently washed and dried using the Tecan HS 4800 hybridization station.

Arrays were scanned in an Agilent DNA microarray scanner. Data of two dye- swapped pairs were normalised and fold-differences in expression was cal culated.

Microarray analysis was performed with Ingenuity Pathways Analysis (Ingenuity systems).

Results

D G K

c

s t i m u l a t e s m y o b l a s t d i f f e r e n t i a t i o n

To determine whether DGKc is involved in differentiation, we used mouse ^C2C12 myoblasts that differentiate into muscle in medium containing low serum. Dif- ferentiation of C2C12 cells is a well characterised process. Within 24 hours of differentiation, cells begin to express myogenin and 36 to 48 hours after stimu-

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lation, late differentiation markers such as myosin heavy chain (MHC) and muscle creatine kinase (MCK) are expressed. Approximately 72 hours after differentiation induction, myoblast cells begin to fuse and to form multinuclear myotubes.

C2C12 cells were retrovirally transduced with wild-type DGKc (wtDGKc), kinase- inactive DGKc (kd^DGKc), or empty vector and after appropriate selection, the stable cell lines were stimulated to differentiate. In wtDGKc overexpressing cells, myotube formation was evident after only three days of differentiation (Fig. 1). In control cells or cells expressing kdDGKc myotube formation was evident at day four.

Furthermore, at day 5 and 6 of differentiation, wtDGKc overexpressing cells formed larger myotubes compared to the other cell lines. These results suggest that wtDGKc stimulates differentiation of C2C12 cells into muscle.

To test whether DGKc stimulates the expression of muscle-specific genes, we determined changes in the expression of myogenin and MHC by Western blotting.

As shown in Figure 2, expression of myogenin increased early in differentiation and declines after 6 days, whereas MHC increased gradually during C2C12 differentiation. In cells expressing wtDGKc,^MHC expression was higher and occurred more rapidly during the differentiation process compared to vector controls. In contrast, expression of MHC in cells expressing kdDGKc was lower compared to wtDGKc cells Fig. 1.

Overexpression of wild-type DGKc stimulates C2C12 myoblast diff erentiation.

C2C12 cells stably expressing wild-type (wt)DGKc, kinase -inactive (kd)DGKc or

empty vector were induced to differentiate. Microscope pictures are shown of a representative field at different time - points of differentiation.

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and their maximal induction is slower than the vector control or wtDGKc cells. Myo- genin expression was enhanced in wtDGKc expressing cells early in differentiation, but expression was comparable in all cell lines at 5 days of differentiation.

These data suggest that overexpression of DGKc increases the levels of muscle- specific proteins.

M i c r o a r r a y a n a l y s i s : D G K

c

u p r e g u l a t e s e x p r e s s i o n o f m a n y m u s c l e - s p e c i f i c g e n e s

To investigate how overexpression of DGKc regulates myogenesis, we compared gene expression profiles in 5 day differentiated C2C12 cells overexpressing wtDGKc with vector control cells using microarray analysis. A list of upregulated genes is shown in Table 1. Myogenin, MCK and myosin polypeptide chains (myosin heavy chain 2A, myosin light chain 1, and myosin regulating light chain) expression was increased in DGKc overexpressing cells. Analysis of the microarray data with In- genuity pathway analysis showed most significant increases in expression of genes involved in muscle contraction, including all components of the troponin complex (troponin C, troponin I, and troponin T), tropomyosin, myomesin 2, myosin-binding protein H, and genes involved in Ca²⁺-homeostasis (calsequestrins, Ryanodine receptor 1, and Ca²⁺-transporting ATPase). In addition, expression of muscle-specific signalling proteins was increased in DGKc overexpressing cells, including insulin signalling, focal adhesion signalling and signalling proteins located in the Fig. 2.

Early induction of MHC and myogenin is enhanced in wtDGKc expressing C2C12 myoblasts during diff erentiation.

Differentiating C2C12 cells overexpressing wtDGKc, kdDGKc, or vector were lysed at different time -points of differentiation and analysed by Western

blotting. Expression of muscle - specific markers is shown in the left panels, whereasDGKc expression and loading controls are shown in the right panels.

All panels are derived from seq uential staining of the same blot.

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muscle development,

cy tosk eleton a nd contr a cti on A ccessi on f old- p- va lue f uncti on C ontr a cti on M y oD ta r g et

increase

MYOGLOBIN NM_ 013593.2 3,38 8,71E-14 oxygen storage

ERYTHROCYTE

TROPOMODULIN (E-TMOD) (TROPOMODULIN 1)

NM_ 021883.1 2,49 1,37E-30 muscle development

MYOGENIN (MYOD1-RELATED

PROTEIN) NM_ 031189.1 2,00 1,42E-9 muscle development

MYOSIN, HEAVY POLYPEPTIDE 2, SKELETAL MUSCLE, ADULT;

MYOSIN HEAVY CHAIN 2A

NM_ 030679.1 5,71 2,51E-11 cytoskeleton

MYOSIN LIGHT CHAIN 1, SKELETAL MUSCLE ISOFORM (MLC1F) (A1 CATALYTIC) (ALKALI)

NM_ 021285.1 3,21 5,86E-13 cytoskeleton

MYOSIN REGULATORY LIGHT CHAIN 2, SKELETAL MUSCLE ISOFORM, FAST (MLC2F)

NM_ 016754.3 2,92 1,31E-21 cytoskeleton MyoD

ACTIN, ALPHA SKELETAL

MUSCLE (ALPHA-ACTIN 1) NM_ 009606.1 2,08 0,001 cytoskeleton MyoD

MYOSIN LIGHT CHAIN 1, ATRIAL/FETAL ISOFORM (MLC1A) (MLC1EMB)

NM_ 010858.3 3,03 2,93E-23 contraction

TROPONIN I, FAST SKELETAL MUSCLE (TROPONIN I, FAST-TWITCH ISOFORM)

NM_ 009405.1 3,01 1,43E-32 contraction MyoD

TROPONIN C2, SKELETAL

MUSCLE, FAST (STNC) NM_ 009394.2 3,01 6,70E-32 contraction x MyoD

TROPONIN I, SKELETAL,

SLOW 1 NM_ 021467.4 2,90 4,52E-27 contraction MyoD

TROPOMYOSIN ALPHA 3 CHAIN (TROPOMYOSIN 3)

(TROPOMYOSIN GAMMA)

P21107 2,88 1,37E-6 contraction

TROPONIN T3, SKELETAL, FAST;

SKELETAL MUSCLE FAST- TWITCH TNT

NM_ 011620.1 2,44 2,62E-30 contraction x MyoD

MYOSIN-BINDING PROTEIN H

(MYBP-H) (H-PROTEIN) NM_ 016749.2 2,31 1,40E-13 contraction x MyoD

TROPONIN T2, CARDIAC

MUSCLE ISOFORMS (TNTC) NM_ 011619.1 2,15 7,14E-7 contraction x MyoD

TROPONIN T1, SKELETAL, SLOW; SKELETAL MUSCLE SLOW-TWITCH TNT

NM_ 011618.1 2,13 4,55E-27 contraction x

T a b le 1 . Microarray-derived list of genes that are upregulated in C2C12 cells overexpressing DGKc compared to vector controls that were diff erentiated for 5 days. Fold-increases in expression relative to vector controls and p-values are shown. MyoD target genes and genes involved in contraction as indicated by Ingenuity, and global functions of genes are shown in the right columns.

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muscle development,

cytoskeleton and contraction Accession fold- p-value function Contraction MyoD target

TROPONIN C, SLOW SKELETAL AND CARDIAC MUSCLES (TN-C)

NM_009393.1 2,13 8,81E-8 contraction MyoD

MYOMESIN 2; SKELETAL

MUSCLE 165KD PROTEIN NM_008664.1 1,78 1,46E-14 contraction x

HISTIDINE RICH CALCIUM

BINDING PROTEIN NM_010473.1 1,76 4,47E-7 contraction x

ALPHA-ACTININ 2 (ALPHA ACTININ SKELETAL MUSCLE ISOFORM 2)

NM_033268.3 2,59 2,91E-5 actin cross-linking x

ALPHA-ACTININ 3 (ALPHA ACTININ SKELETAL MUSCLE ISOFORM 3)

NM_013456.1 1,87 1,85E-8 actin cross-linking x

S ignalling

NOV PROTEIN HOMOLOG PRECURSOR (NOVH) (NEPHROBLASTOMA OVEREXPRESSED GENE PROTEIN HOMOLOG)

NM_010930.3 2,74 3,60E-14 signalling/formation of myotubes

SERINE/THREONINE PROTEIN KINASE 23 (EC 2.7.1.37) (MUSCLE-SPECIFIC SERINE KINASE 1) (MSSK-1)

NM_019684.1 2,40 2,08E-15 signalling

MUSCLE CREATINE KINASE

(EC 2.7.3.2) (MCK) NM_007710.1 2,38 1,19E-5 signalling/activation

of muscle MyoD

ECTONUCLEOTIDE PYROPHOSPHATASE/

PHOSPHODIESTERASE 2 (AUTOTAXIN)

NM_015744.1 2,04 3,65E-6 signalling MyoD

STRIATED MUSCLE ACTIVATOR OF

RHO-DEPENDENT SIGNALING

NM_175456.2 1,79 3,05E-6 signalling

MYOZ ENIN-LIKE 2;

CALSARCIN-1 NM_021503.1 3,90 2,40E-20 Z -disk signalling

LIM DOMAIN PROTEIN, CARDIAC (MUSCLE LIM PROTEIN) (CYSTEINE-RICH PROTEIN 3) (CRP3)

NM_013808.3 2,85 6,35E-17 Z -disk signalling/

muscle development

LIM DOMAIN BINDING 3;

Z -BAND ALTERNATIVELY SPLICED PDZ -MOTIF PROTEIN;

CYPHER 1; CYPHER 2

NM_011918 2,61 4,72E-29 Z -disk signalling

DIABETES RELATED ANKYRIN REPEAT PROTEIN

ANKYRIN REPEAT DOMAIN PROTEIN 2 (SKELETAL MUSCLE

NM_153502.2 2,12 6,78E-11 Z -disk signalling Table 1. (continued)

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muscle development,

cytoskeleton and contraction Accession fold- p-value function Contraction MyoD target

ANKYRIN REPEAT PROTEIN)

(MARPP) NM_020033.1 1,97 0,000 Z disk signalling

ACETYLCHOLINE RECEPTOR PROTEIN, GAMMA CHAIN PRECURSOR

NM_009604.2 2,01 2,81E-16 synaptic

transmission x

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 PRECURSOR (IGFBP-3) (IBP-3) (IGF-BINDING PROTEIN 3)

NM_008343.1 2,18 5,35E-26

insulin signalling/

diff erentiation of myoblasts

INSULIN-LIKE GROWTH FACTOR II PRECURSOR (MULTIPLICATION

STIMULATING POLYPEPTIDE) (IGF-II)

NM_010514.2 1,77 0,009

insulin signalling/

diff erentiation of myoblasts

MyoD indirect

SMALL MUSCLE PROTEIN, X-LINKED; CARDIAC AND SKELETAL MUSCLE PROTEIN;

CHISEL.

NM_025357.1 3,17 5,44E-13 focal adhesion

signalling x

INTEGRIN BETA 1 BINDING

PROTEIN 2 (MELUSIN) NM_013712.1 1,90 1,53E-23

focal adhesion signalling/muscle development Ca h omeostasis

CALSEQUESTRIN, SKELETAL MUSCLE ISOFORM PRECURSOR (CALSEQUESTRIN 1)

NM_009813.1 3,09 0,001 storage/release of

Ca x

CALSEQUESTRIN, CARDIAC MUSCLE ISOFORM PRECURSOR (CALSEQUESTRIN 2)

NM_009814.1 2,98 1,35E-12 storage/release of

Ca x

RYANODINE RECEPTOR 1, SKELETAL MUSCLE (RYANODINE RECEPTOR TYPE 1)

NM_009109.1 2,29 2,39E-7 release of Ca x MyoD

CALCIUM-TRANSPORTING ATPASE, CARDIAC MUSCLE, FAST-TWITCH 1

NM_007504.2 2,01 1,87E-8 Ca homeostasis x

Z disk of muscle, including calsarcin 1, LIMdomain binding 3, and ankyrin repeat domain protein 2. The Z disk, where actin filaments of neighbouring sarcomeres overlap and are cross-linked by _-actinin, contains signalling complexes that are involved in sensing (mechanical) stress (Hoshijima, 2006). Network analysis of the microarray data revealed that many of the genes that are upregulated are direct targets of the muscle-specific gene transcriptional activator MyoD.

Table 1. (continued)

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R e g u l a t i o n a n d q u a n t i t a t i o n o f D G K

c

- e n h a n c e d g e n e e x p r e s s i o n To confirm data obtained with the microarray analysis, real-time PCR was used to measure the mRNA levels of myogenin and MCK, two genes shown to be upregulated during differentiation by overexpression of DGKc. The fold-difference in m^RNA expression normalised to a house keeping gene and relative to undifferentiated vector controls was calculated. Whereas myogenin mRNA expression was comparable in nondifferentiated cells, significantly higher myogenin mRNA levels (2.5-fold) were observed in wtDGKc overexpressing cells compared to vector controls (Table 2).MCK mRNA levels were also significantly higher in cells expressing wtDGKc compared to vector controls at 5 days of differentiation (2-fold). In order to determine if the activity of DGKc is required to stimulate the upregulation of MCK or myogenin mRNA, we overexpressed kdDGKc. This mutant was expressed to comparable levels as the wild-type enzyme; however, it was unable to increase myogenin and MCK mRNA expression and expression of myogenin was even lower in kdDGKc expressing cells compared to vector controls. We also assessed whether DGKc levels are regulated during differentiation. No significant changes in DGKc mRNA levels were detected during the differentiation process, as revealed by

Days of differentiation

0 3 5 7

myogenin wtDGKc 0.77

(0.72-0.82)

23.26 * (21.06-25.70)

66.37 * * (61.49-71.65)

35.14 * (32.23-38.31) kdDGKc 0.56 *

(0.54-0.59)

9.25 (8.41-10.19)

19.29 * (17.35-21.46)

22.09 (19.65-24.83)

vector 1.00

(0.90-1.11)

10.65 (9.69-11.69)

24.76 (22.61-27.11)

25.95 (24.14-27.89)

MCK wtDGKc 0.83

(0.63-1.09)

67.77 (43.05-106.68)

329.13 * * (270.20-400.90)

321.80 (226.22-457.75)

kdDGKc 0.52

(0.32-0.82)

74.16 (57.22-96.11)

153.81 (114.46-206.69)

202.60 (162.08-253.26)

vector 1.00

(0.75-1.33)

91.30 (72.99-114.19)

180.71 (141.79-230.31)

222.09 (172.38-286.13)

Days of differentiation

0 3 5 7

Vector 1.00

(0.83-1.20)

0.83 (0.64-1.08)

0.73 (0.62-0.86)

1.04 (0.90-1.21) Table 2 . Fold-diff erence in myogenin and MCK mRNA expression relative to undiff erentiated vector control cells.

RNA was isolated from C2C12 cells stably expressing wtDGKc or vector at diff erent time-points of diff erentiation and quantified using real-time PCR. Significance: * , p < 0.05; * * , p < 0.01 (Student’s t-test).

Table 3 . Fold-diff erence in DGKc expression relative to undiff erentiated cells. RNA was isolated from C2C12 cells at diff erent time-points of diff erentiation and quantified using real-time PCR. No significant changes were found.

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real-time PCR (Table 3). These results confirm that active DGKc enhances differentiation of C2C12 cells through enhancing transcriptional regulation of muscle- specific genes.

An upregulation of the mRNA encoding myogenin and MCK may occur either as a consequence of reduced degradation or increased synthesis through the activation of myogenic regulating factors (MRF). To test if DGKc increases MRF activity, we used the promoter regions of myogenin and MCK, two known targets of MRF activity, linked to the expression of luciferase. We also tested if DGKc could also stimulate transcription from a synthetic promoter containing four repeated E box sites (4RE-luc) that binds MRFs including MyoD (Puri et al., 2001). As a control we utilised the promoter for p21, since p21, although upregulated during differentiation, is not a direct target for MyoD transcriptional activity. Stable C2C12 cell lines expressing wtDGKc or empty vector were transfected with reporter constructs, differentiated for two days and analysed for luciferase activity. Luciferase expression driven from the promoters of myogenin and MCK and from the synthetic promoter was significantly increased in C2C12 cells overexpressing wtDGKc compared to empty vector (Fig. 3). In contrast, luciferase expression driven by the control p21 promoter was not significantly regulated by DGKc expression. These results suggest that DGKc stimulates MRF (MyoD)-dependent transcriptional activity, which may explain our finding of enhanced levels of the muscle-specific proteins, MHC and myogenin (Fig. 2).

R o l e o f pR B i n m u s c l e d i f f e r e n t i a t i o n

The retinoblastoma protein (pRB) is a well established regulator of myogenesis (De Falco et al., 2006). Deletion of pRB in mice leads to profound skeletal muscle defects and, in vitro,MEFs that are deficient in pRB are unable to differentiate (Novitch et al., 1996). Both in mice and in in vitro differentiation assays the profound defect in muscle differentiation induced by the deletion of pRB can be partially overcome through inhibition of Ras activity (Lee et al., 1999; Takahashi et al., 2004).

DGKc is a regulator of Ras activation as it can suppress the levels of cellular DAG which can increase the activity of an upstream Ras activator, Ras-GRP (Topham and Prescott, 2001). Furthermore, we have previously demonstrated that DGKc activity is potently stimulated by its interaction with pRB and that overexpression of DGKc is able to partially reconstitute a cell cycle checkpoint lost in pRB-deplet- ed cells (Los et al., 2006). We therefore assessed if overexpressed DGKc could reconstitute (through inhibition of Ras) differentiation in C2C12 cells that lack a functional pRB.

C2C12 myoblasts were transduced with human papilloma virus E7 oncoprotein that targets pRB for degradation. As expected, wild-type cells differentiated, however E7 expressing cells were unable to differentiate (Fig. 4). We then gener- ated C2C12 cells expressing both E7 and DGKc.DGKc was expressed during the differentiation protocol; however, overexpression of DGKc did not alleviate the defect in differentiation induced by the lack of pRB. As pRB-negative C2C12 cells do not differentiate, we were unable to determine whether enhanced C2C12 myoblast differentiation in DGKc overexpressing cells is dependent on pRB.

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D G K

c

- n u l lM E Fs a r e u n a b l e t o d i f f e r e n t i a t e i n t o m u s c l e

Since overexpression of DGKc stimulates muscle differentiation, we tested whether muscle differentiation is inhibited in DGKc-null cells. Murine embryonic fibroblasts (MEFs) were isolated from mice that were either wild-type or nullizygous with respect to DGKc. The MEFs were transduced with a retroviral construct encoding MyoD and differentiated in DMEM containing 2% horse serum and insulin. At different time-points of differentiation expression of muscle-specific proteins was assessed by Western blotting. Whereas wild-type MEFs differentiated and express MHC,DGKc^-/-MEFs were unable to induce the expression of MHC (Fig. 5). Myogenin expression was very low and difficult to detect, but was only induced in wild-type MEFs (not shown). These results indicate that DGKc is required for differentiation of MEFs into myoblasts.

Discussion

We have shown that overexpression of DGKc stimulates ^C2C12 myoblast differentiation and that stimulation of differentiation requires the activity of DGKc. Over- expression of DGKc induced enhanced muscle-specific gene expression, including Fig. 3.

DGKc stimulates promoter activity of muscle-specific genes.

C2C12 cells overexpressing wtDGKc or vector were transfected with MCK-, myogenin-, or 4RE-luciferase constructs.

Control cells (0 days) and cells that were induced to differentiate for two

days, were lysed and luciferase activity was measured. Firefly luciferase activity was corrected for Renilla luciferase activity and plotted in the histograms shown as means ± S.E. (n= 3). Signifi- cance: * , p < 0.05 (Student’s t-test).

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genes involved in muscle contraction and signalling. Increased expression of some of the muscle-specific genes occurs as a consequence of the enhancement of MRF activity, as DGKc overexpression stimulated luciferase expression from MRF driven promoters. Finally, and most importantly, MEFs isolated from DGKc knock-out mice were unable to differentiate into muscle.

Microarray analysis revealed that overexpression of DGKc significantly stimulated the expression of cytoskeletal and signalling genes as well as genes involved in muscle contraction. Both slow-twitch and fast-twitch muscle genes became expressed, indicating that DGKc did not just stimulate differentiation to a specific muscle type (for a review concerning different muscle types, see (Zierath and Hawley, 2004)).

Our results suggest that DGKc may enhance muscle-specific gene expression by regulating their transcription, as DGKc stimulates luciferase expression driven by the myogenin- and MCK promoters. Furthermore, it is likely that MyoD is one of the transcription factor targets for DGKc, as luciferase expression driven by the synthetic promoter with four repeated E-box sites (4RE-luc) was also enhanced by DGKc overexpression. In addition, MyoD stimulates promoter activity

Fig. 4.

DGKc is unable to reconstitute diff erentiation of pRB-negative C2C12 cells.

C2C12 cells stably expressing DGKc and/or human papilloma virus E7 or their respective empty vectors (indicated), were differentiated and lysed at different time -points. L ysates were separated on SDS-PAGE, blotted to nitrocellulose and stained for myosin, DGKc and myogenin. Autoradiographs of equal exposure times are shown for each staining.

Fig. 5.

DGKc^-/-MEFs f ail to diff erentiate into muscle and are unable to express MHC. DGKc^-/-MEFs and wild-type MEFs stably expressingMyoD were stimulated to differentiate and lysed at different time -points of differentiation. L ysates were analysed by Western blotting and stained for myosin and DGKc.

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of myogenin and MCK (Puri et al., 2001; Berkes and Tapscott, 2005), which may, therefore, be the mechanism by which DGKc affects myogenin and ^MCK promoter activity. Furthermore, from our microarray analysis it appears that many of the genes upregulated by the overexpression of DGKc are direct targets for MyoD transcriptional activity. As a negative control, luciferase expression driven by the p21 promoter, being no target for MyoD, was not significantly stimulated in DGKc overexpressing cells. Therefore, the activation of a specific set of promoters suggests that DGKc overexpression targets specific aspects of the muscle differentiation programme.

How DGKc regulates differentiation-specific promoter activity needs to be further investigated. DGKc may facilitate co-regulation of MyoD with other transcription factors, including MEF2 to stimulate promoter activity. For example, the MCK promoter contains E-box and MEF2 sites that synergistically stimulate MCK transcription (Molkentin et al., 1995; Puri et al., 2001). Alternatively, DGKc may regulate the recruitment of transcription factor co-activators and co-repressors to stimulate muscle-specific gene expression, including histone modifying proteins such as acetylases/deacetylases and methylases, or proteins of the SWI/SNF chromatin remodelling complex (Sartorelli and Caretti, 2005).

Although DGKc fascilitates muscle differentiation, its mRNA levels did not significantly change during the differentiation process. A study where gene targets of MyoD, myogenin and MEF2 were identified in a large-scale assay revealed that the DGKc promoter was not a target for these transcription factors (Blais et al., 2005). Therefore, during differentiation DGKc may be regulated at the (post)translational level and/or through regulation of its activity.

Our finding that DGK activity is required to stimulate C2C12 differentiation, implies either the removal of DAG and/or the production of PA.DAG activates PKC, a common downstream target of many growth factors, including Fibroblast growth factor-2 (FGF-2), that can inhibit myogenesis. PKC can phosphorylate myogenin, MyoD and MRF4 and phosphorylation of these transcription factors inhibits interaction with E-box proteins, DNA-binding and transcriptional activation in vitro (Li et al., 1992; Liu et al., 1998; Hardy et al., 1993). Interestingly, a MyoD mutant in which a PKC phosphorylation site was mutated was able to differentiate in the presence of serum (Liu et al., 1998). DGKc may also stimulate differentia - tion by inhibiting DAG-mediated Ras-GRP activation (Topham and Prescott, 2001).

DAG activates Ras-GRP, which in turn stimulates loading of GTP onto Ras. Over- expression of DGKc has been shown to decrease the levels of Ras-GTP, and Ras-GTP levels are upregulated in T cells derived from mice in which the gene encoding DGKc has been deleted (Zhong et al., 2003). Active Ras inhibits C2C12 differen tiation, through the activation of the MAPK pathway (Conejo et al., 2002).

Interestingly, functional inactivation of pRB also leads to Ras activation and prevents MyoD dependent myogenesis (Lee et al., 1999). I n vivo and in vitro the defect in muscle differentiation induced by the loss of pRB is partially restored by inhibition of Ras activity (Lee et al., 1999; Takahashi et al., 2004). However, we were unable to restore differentiation in C2C12 cells lacking a functional pRB by solely the overexpression of DGKc. This may suggest that in C2C12 cells Ras-

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GRP pathway is not a major regulator of Ras activation. Alternatively, E7 which we used to functionally inactivate pRB, might inhibit myogenesis by multiple mechanisms.

There is also the possibility that the product of DGKc,PA, stimulates the differentiation. While it is still unclear how PA mediates its second messenger effects some target enzymes for this lipid, for example mTOR, have been suggest- ed (Avila-Flores et al., 2005). mTOR also promotes muscle differentiation of C2C12 cells, although the exact mechanism is poorly understood (Ohanna et al., 2005).

mTOR activity also promotes transcription of insulin-like growth factor (IGF-II) that, after being secreted, activates IGF signalling that stimulates differentiation via the PI3K-Akt pathway (Erbay et al., 2003). Interestingly, our microarray data showed increased IGF-II precursor protein expression in C2C12 cells overexpressing wtDGKc that could be explained by ^DGKc-mediated ^PA production and mTOR activation.

The requirement of DGKc for muscle differentiation seems obvious from the remarkable finding that DGKc^-/-MEFs were unable to differentiate into muscle.

However, this is not in agreement with the phenotype of DGKc^-/- mice that did not show gross abnormalities in skeletal muscle development (G. Koretzky, personal communication). Differentiation of MEFs in vitro, however, is only dependent on the presence of insulin, while differentiation in vivo is much more complicated requiring multiple signals including those from the Wnt and sonic hedgehog pathways (reviewed in Cossu et al., 2000). Thus, it would be interesting to test the hypothesis that defects in insulin signalling may lead to impaired differentiation of DGKc^-/-MEFs in vitro, whereas, in vivo, additional mechanism(s) may compen- sate this defect to allow proper muscle differentiation. Furthermore, it is clear from our microarray data that many of the genes that are upregulated are those involved in contractile responses and therefore a detailed histological exami- nation of the skeletal muscle, muscular movement, and muscular function may yet unveil a specific muscular phenotype of the DGKc knock-out mice.

In addition to the observed role of DGKc in muscle development, DGKc also appears to play an important functional role in skeletal muscle function. DGKc is localised at the sarcolemma where it forms a complex with syntrophins and dystrophins to regulate actin dynamics (Abramovici et al., 2003). Disruption of DGKc binding to syntrophins led to the localisation of DGKc in the nucleus and abnormal cytoskeletal morphology. Muscle fibres derived from a mouse model of Duchenne muscular dystrophy, containing a point-mutation in dystrophin, also show defects in cytoskeletal organisation and a decrease in DGKc expres - sion and mislocalisation of DGKc in the nucleus. Furthermore, in regenerating muscle fibers of m d x mice, DGKc and syntrophins were upregulated. Since it is thought that regenerating fibers upregulate a similar myogenic program to that induced in developing muscle (Palacios and Puri, 2006), DGKc may also be im portant during muscle regeneration in adult tissues.

Note added: After finishing our experi mental work, a paper of Evangelisti et al (Evangelisti et al., 2006) appeared in which DGKc downregulation by siRNA markedly impaired myogenic differentiation of C2C12 cells, in agreement with our data.

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