Knockdown of MTP18, a Novel Phosphatidylinositol 3-Kinase-dependent Protein, Affects Mitochondrial Morphology and Induces Apoptosis

We identified a novel human cDNA encoding a mitochondrial protein, MTP18 (mitochondrial protein, 18 kDa) as a transcriptional downstream target of phosphatidylinositol (PI) 3-kinase signaling. We demonstrate that MTP18 mRNA as well as protein expression is dependent on PI 3-kinase activity. Confocal microscopy and biochemical fractionation revealed a mitochondrial localization of MTP18. Loss-of-function analysis employing antisense molecules revealed that MTP18 is essential for cell viability in PC-3 and HaCaT cells. We show that knockdown of MTP18 protein level results in a cytochrome c release from mitochondria and consequently leads to apoptosis. In addition, HaCaT cells with reduced levels of MTP18 become more sensitive to apoptotic stimuli. This effect is accompanied by dramatic subcellular alterations. Reduction of MTP18 impairs mitochondrial morphology resulting in the formation of a highly interconnected mitochondrial reticulum in COS-7 cells. Conversely, overexpression of MTP18 induces a punctuate morphology of mitochondria suggesting also a functional role of MTP18 in maintaining the mitochondrial integrity. Hence, our data indicate an unexpected connection of PI 3-kinase signaling, apoptosis and the morphology of mammalian mitochondria. We identified a novel human cDNA encoding a mitochondrial protein, MTP18 (mitochondrial protein, 18 kDa) as a transcriptional downstream target of phosphatidylinositol (PI) 3-kinase signaling. We demonstrate that MTP18 mRNA as well as protein expression is dependent on PI 3-kinase activity. Confocal microscopy and biochemical fractionation revealed a mitochondrial localization of MTP18. Loss-of-function analysis employing antisense molecules revealed that MTP18 is essential for cell viability in PC-3 and HaCaT cells. We show that knockdown of MTP18 protein level results in a cytochrome c release from mitochondria and consequently leads to apoptosis. In addition, HaCaT cells with reduced levels of MTP18 become more sensitive to apoptotic stimuli. This effect is accompanied by dramatic subcellular alterations. Reduction of MTP18 impairs mitochondrial morphology resulting in the formation of a highly interconnected mitochondrial reticulum in COS-7 cells. Conversely, overexpression of MTP18 induces a punctuate morphology of mitochondria suggesting also a functional role of MTP18 in maintaining the mitochondrial integrity. Hence, our data indicate an unexpected connection of PI 3-kinase signaling, apoptosis and the morphology of mammalian mitochondria. The phosphatidylinositol (PI) 1The abbreviations used are: PI, phosphatidylinositol; TNFα, tumor necrosis factor α; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; GFP, green fluorescent protein; GB, GeneBloc; MM, mismatch; RT, reverse transcriptase; PARP, poly(ADP-ribose) polymerase. 1The abbreviations used are: PI, phosphatidylinositol; TNFα, tumor necrosis factor α; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; GFP, green fluorescent protein; GB, GeneBloc; MM, mismatch; RT, reverse transcriptase; PARP, poly(ADP-ribose) polymerase. 3-kinase pathway has been extensively studied for its role in regulating cell growth, development, motility, adhesion, glucose transport, immune response, and survival (1Katso R. Okkenhaug K. Ahmadi K. White S. Timms J. Waterfield M.D. Annu. Rev. Cell Dev. Biol. 2001; 17: 615-675Google Scholar). A chronic activation of the PI 3-kinase pathway contributes to tumorigenesis and metastasis (2Luo J. Manning B.D. Cantley L.C. Cancer Cell. 2003; 4: 257-262Google Scholar). Activation of PI 3-kinase signaling and its downstream effector, the protein-serine/threonine kinase Akt, is considered to be one major signaling pathway by which survival factors prevent apoptosis (3Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Google Scholar, 4Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Google Scholar). Some reports suggest that chronically activated PI 3-kinase can protect tumor cells against various apoptotic stimuli (3Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Google Scholar). The activity of members of the Bcl-2 family is regulated by the PI 3-kinase downstream effector, the protein-serine/threonine kinase Akt. Hyperactivation of Akt inhibits the pro-apoptotic activities of Bad, Bax, and Bid (5del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Google Scholar, 6Tsuruta F. Masuyama N. Gotoh Y. J. Biol. Chem. 2002; 277: 14040-14047Google Scholar, 7Majewski N. Nogueira V. Robey R.B. Hay N. Mol. Cell. Biol. 2004; 24: 730-740Google Scholar). Akt has also been reported to inhibit apoptosis by phosphorylation of several other proteins (8Wymann M.P. Zvelebil M. Laffargue M. Trends Pharmacol. Sci. 2003; 24: 366-376Google Scholar). However, it is possible that besides Akt other additional effectors downstream of PI 3-kinase exist which are responsible for mediating cell survival. For instance, it is unknown whether besides controlling the activity of the cell death machinery the expression of other integral mitochondrial specific proteins is directly dependent on PI 3-kinase signaling. The release of several proapoptotic mitochondrial proteins from mitochondrial intermembrane space into the cytoplasm has been reported to be crucial for the onset of apoptosis (9Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Google Scholar). In addition, recent reports point to a change in mitochondrial morphology as a prerequisite for the release of these mitochondrial factors (10Karbowski M. Youle R.J. Cell Death Differ. 2003; 10: 870-880Google Scholar, 11Frank S. Gaume B. Bergmann-Leitner E.S. Leitner W.W. Robert E.G. Catez F. Smith C.L. Youle R.J. Dev. Cell. 2001; 1: 515-525Google Scholar). Even though the mitochondrial disintegration during the executing steps of apoptosis is established, it seems that the maintenance of the mitochondrial morphology is also important to ensure mitochondrial function and homeostasis. For example mitochondrial dysfunction itself has been linked to various diseases and cancer (12Greenamyre J.T. MacKenzie G. Peng T.I. Stephans S.E. Biochem. Soc. Symp. 1999; 66: 85-97Google Scholar, 13Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. Grosgeorge J. Turc-Carel C. Perret E. Astarie-Dequeker C. Lasquellec L. Arnaud B. Ducommun B. Kaplan J. Hamel C.P. Nat. Genet. 2000; 26: 207-210Google Scholar, 14Weiss J.N. Korge P. Honda H.M. Ping P. Circ. Res. 2003; 93: 292-301Google Scholar). However, despite of the identification of several proteins involved in maintaining mitochondrial morphology the regulation or the participating signal transduction pathways for these processes are still unknown. Here we present the identification and characterization of a previously unknown novel downstream effector of PI 3-kinase signaling. The PI 3-kinase-dependent expression is demonstrated on mRNA and protein level. We show that this novel protein MTP18 (mitochondrial protein, 18 kDa) localized to mitochondria of mammalian cells. Loss-of-function studies using MTP18-specific antisense molecules reveal that this mitochondrial protein is essential for cell viability. Moreover, reduction of the MTP18 protein level induces the release of cytochrome c, activates the caspase cascade, and leads to cell death. Overexpression as well as loss-of-function experiments suggest a functional role of MTP18 in maintaining the integrity of the mitochondrial network. Cell Culture—Human prostate carcinoma PC-3, HeLa, and COS-7 cells (American Type Culture Collection) were cultured as described (15Czauderna F. Fechtner M. Aygun H. Arnold W. Klippel A. Giese K. Kaufmann J. Nucleic Acids Res. 2003; 31: 670-682Google Scholar, 16Klippel A. Escobedo M.A. Wachowicz M.S. Apell G. Brown T.W. Giedlin M.A. Kavanaugh W.M. Williams L.T. Mol. Cell. Biol. 1998; 18: 5699-5711Google Scholar, 17Sternberger M. Schmiedeknecht A. Kretschmer A. Gebhardt F. Leenders F. Czauderna F. Von Carlowitz I. Engle M. Giese K. Beigelman L. Klippel A. Antisense Nucleic Acid Drug Dev. 2002; 12: 131-143Google Scholar). Human keratinocytes (HaCaT) (18Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Google Scholar) were obtained from P. Boukamp (Deutsches Krebsforschungszentrum, Germany) and cultured as described (19Kretschmer A. Moepert K. Dames S. Sternberger M. Kaufmann J. Klippel A. Oncogene. 2003; 22: 6748-6763Google Scholar). GeneBloc transfections were carried out as desribed (17Sternberger M. Schmiedeknecht A. Kretschmer A. Gebhardt F. Leenders F. Czauderna F. Von Carlowitz I. Engle M. Giese K. Beigelman L. Klippel A. Antisense Nucleic Acid Drug Dev. 2002; 12: 131-143Google Scholar). MTP18 expression plasmids were transfected using Effectene™ (Qiagen) according to the manufacturer's instructions. Cell proliferation was measured using WST-1 reagent (Roche Applied Science). PC-3 cells (5 × 103) in 200 μl of culture medium were seeded into 96-well plates. After incubation for 24 h, the cells were transfected with GeneBlocs or treated with LY294002 (day 0). At the end of treatment, 10:1 of WST-1 reagent were added into wells and incubated for 1 h and measured according to the manufacturer's protocol using a SpectraMax 190 (Molecular Devices). Cell growth on matrigel matrix (BD Biosciences) was assayed according to Sternberger et al. (17Sternberger M. Schmiedeknecht A. Kretschmer A. Gebhardt F. Leenders F. Czauderna F. Von Carlowitz I. Engle M. Giese K. Beigelman L. Klippel A. Antisense Nucleic Acid Drug Dev. 2002; 12: 131-143Google Scholar) using PC-3 cells (75,000 cells per well). To induce apoptosis, HaCaT cells (1 × 106 in 10-cm plates) were kept in 5 ml of phosphate-buffered saline and exposed to 10 or 50 mJ/cm2 using the UVB 500 UV cross-linker (Hoefer Scientific Instruments). After radiation, phosphate-buffered saline was replaced by 10 ml of fresh medium. For TNFα-induced apoptosis, cells were incubated with 5 or 10 ng/ml TNFα (+10 μg/ml cycloheximide) with for 2 h. FACS analysis was performed as described (19Kretschmer A. Moepert K. Dames S. Sternberger M. Kaufmann J. Klippel A. Oncogene. 2003; 22: 6748-6763Google Scholar). Immunoblotting and Antibodies—The preparation of cell extracts and immunoblot analysis was carried out as described (16Klippel A. Escobedo M.A. Wachowicz M.S. Apell G. Brown T.W. Giedlin M.A. Kavanaugh W.M. Williams L.T. Mol. Cell. Biol. 1998; 18: 5699-5711Google Scholar). Isolation of a highly enriched mitochondrial fraction from the cytosolic fraction of PC-3 cells was performed using a mitochondria/cytosol fractionation kit (Active Motif). The determination of mitochondrial cytochrome c release was analyzed using a digitonin-based subcellular fractionation technique as described in Takasawa and Tanuma (47Takasawa R. Tanuma M.T. Apoptosis. 2003; 8: 291-299Google Scholar). Polyclonal antibodies against full-length MTP18 were generated by immunizing rabbits with recombinant MTP18 protein produced in Escherichia coli using pET19-b expression vector (Merck Biosciences GmbH). The murine monoclonal antibodies anti-p110α and anti-p85 have been described (16Klippel A. Escobedo M.A. Wachowicz M.S. Apell G. Brown T.W. Giedlin M.A. Kavanaugh W.M. Williams L.T. Mol. Cell. Biol. 1998; 18: 5699-5711Google Scholar, 20Klippel A. Escobedo J.A. Hirano M. Williams L.T. Mol. Cell. Biol. 1994; 14: 2675-2685Google Scholar). Rabbit polyclonal anti-Akt and antiphospho-Akt (Ser-473) antibodies, as well as polyclonal antibodies specific for cleaved poly(ADP-ribose) polymerase (PARP) were obtained from Cell Signaling Technology. The murine monoclonal anti-Hsp60, monoclonal anti-Drp1/DLP1 antibody, and monoclonal anti-cytochrome c antibody (clone 6H2.B4) were purchased from BD Transduction Laboratories. The rabbit polyclonal anti-ETS-1 and p110β antibodies were obtained from Santa Cruz Biotechnology. Plasmids and Antisense Oligonucleotides (GeneBlocs)—The nucleotide sequence of human MTP18 cDNA is deposited under GenBank™ accession number AAH46132. The coding regions of the MTP18 gene was amplified by PCR using gene-specific primers and the Expand Long Template PCR System kit (Roche Applied Science). The amplified product was inserted into the pCR4-TOPO cloning vector (Invitrogen). HA- and MYC-tagged MTP18 expression constructs were generated by subcloning the MTP18 cDNA into pCG-HA (XbaI/BamHI) for N-terminal HA- and pCG-MYC (XbaI/SmaI) for C-terminal MYC fusion, respectively. GFP-MTP18 and MTP18-GFP were generated by PCR and PCR products were subcloned using pcDNA-NT-GFP-TOPO and pcDNA-CT-GFP-TOPO vector system, respectively (Invitrogen). The used GeneBloc (GB) represents the third generation of gapmer antisense oligonucleotides (described in Ref. 17Sternberger M. Schmiedeknecht A. Kretschmer A. Gebhardt F. Leenders F. Czauderna F. Von Carlowitz I. Engle M. Giese K. Beigelman L. Klippel A. Antisense Nucleic Acid Drug Dev. 2002; 12: 131-143Google Scholar). The GBs used in this study have the following sequences: GB p110α, acuccaaAGCCTCTTGcucaguu; mismatch (MM) p110α, acugcaaACCCTGTTGcucacuu; GB p110β, ggcuaacTTCATCTTCcuuccca; MM p110β, ggcuaagTTCTTCATCcuugcca; GB Akt 1, gucuugATGTACTCCccucgu; MM Akt 1, guguugATCTAGTCCccuccu; GB Akt 2, uccuugTACCCAATGaaggag; MM Akt 2, uaguugTAGCCAATCaacgag; GB1 MTP18, gcuucuTGAAGCCTTugucuc; MM1 MTP18, gcaucuAGTACCCATagucuc; GB2 MTP18, agccuuCTTGCCTTTgucaau; MM2 MTP18, agccuaCTTCCCTATgugaau. The respective mismatch positions in the MM control oligomeres are underlined. Northern Blot and RT-PCR (TaqMan)—Human multiple tissue Northern blots were purchased from BD Biosciences Clontech. The blots were hybridized with radiolabeled full-length MTP18 probe (Rediprime II, Amersham Biosciences) according to manufacturer's protocol. Blots were analyzed using a PhosphorImager (Amersham Biosciences). RNA was isolated and purified using the Invisorb spin cell RNA mini kit (InVitek GmbH). Inhibition of MTP18 mRNA expression was detected by multiplex real-time RT-PCR as described previously (19Kretschmer A. Moepert K. Dames S. Sternberger M. Kaufmann J. Klippel A. Oncogene. 2003; 22: 6748-6763Google Scholar). Immunofluorescence Microscopy—About 15–20 h after transfection, cells were fixed for 15 min at room temperature in 4% formaldehyde/phosphate-buffered saline. All subsequent steps for indirect immunofluorescence and MitoTracker Red (Molecular Probes) staining were carried out as described previously (21Santel A. Fuller M.T. J. Cell Sci. 2001; 114: 867-874Google Scholar). Nocodazole (5 μm) treatment was performed as described (22Smirnova E. Griparic L. Shurland D.L. van der Bliek A.M. Mol. Biol. Cell. 2001; 12: 2245-2256Google Scholar). An LSM 510 META confocal microscope (Zeiss) was used for microscopy. Identification of a Novel PI 3-Kinase-dependent Target Gene—To identify novel downstream drug targets in the PI 3-kinase pathway, we analyzed gene expression changes in PTEN–/– PC-3 cells (human prostate cancer cells) after inhibition of PI 3-kinase activity with the small molecule inhibitor LY294002 (23Kaufmann J. Pronk G. Giese K. Klippel A. Biochem. Soc. Trans. 2004; 32: 355-359Google Scholar). The ability of cells to grow on or in extracellular matrix is indicative for the invasive growth potential of cancer cells (23Kaufmann J. Pronk G. Giese K. Klippel A. Biochem. Soc. Trans. 2004; 32: 355-359Google Scholar, 24Bissell M.J. Rizki A. Mian I.S. Curr. Opin. Cell Biol. 2003; 15: 753-762Google Scholar). Since the phenotypic consequences of PI 3-kinase inhibition are more pronounced on cells grown on extracellular matrix when compared with cells grown on plastic, we carried out our gene expression profiling experiments with RNA derived from cells grown on this semisolid surface (Fig. 1A) (23Kaufmann J. Pronk G. Giese K. Klippel A. Biochem. Soc. Trans. 2004; 32: 355-359Google Scholar). The RNA derived from matched samples of PC-3 cells treated with or without the PI 3-kinase-specific inhibitor (LY294002) were used to perform a genome wide gene expression profiling (Affymetrix). This experimental approach led us to the identification of a series of known and novel PI 3-kinase-dependent mRNAs (for details, see Ref. 23Kaufmann J. Pronk G. Giese K. Klippel A. Biochem. Soc. Trans. 2004; 32: 355-359Google Scholar). Here we present the identification and characterization of a novel PI 3-kinase-dependent target gene (GenBank™ accession number AAH46132). According to the DNA chip analysis the mRNA level was reduced in PC-3 cells treated with 10 μm LY294002 when compared with the Me2SO vehicle treated control cells (Fig. 1B, upper panel). To confirm the DNA chip analysis we employed real-time PCR (TaqMan) on independently derived RNA preparations (Fig. 1B, lower panel). With both methods a similar time-dependent decrease of the AAH46132 mRNA was observed. The mRNA level of p110α, one catalytic subunit of PI 3-kinase, was not changed and was used to normalize mRNA amounts in both assays. Having established the PI 3-kinase-dependent mRNA expression we cloned the full-length cDNA of AAH46132 and generated polyclonal antibodies against the recombinant protein. Fig. 2A shows the full-length cDNA including the open reading frame used for recombinant expression of the protein. We named this novel protein MTP18 (mitochondrial protein, 18 kDa) because of the mitochondrial localization and its predicted mass of 18 kDa (see below). A data base search revealed protein homologues in other metazoa (mouse, Caenorhabditis elegans, Drosophila) but not in unicellular organisms like yeast (Fig. 2B). So far none of these homologues has been characterized functionally. Northern blot analysis with AAH46132-specific probes on RNA derived from human tissues revealed a single 1.4-kb-long mRNA. The size is consistent with the full-length construct and suggests the absence of splice variants (Fig. 2C). The strongest expression level was observed in heart and skeletal muscle but transcripts were also detected in other tissues including prostate, trachea, pancreas, kidney, and liver. Lower mRNA expression was detected in brain, lung, spinal cord, bone marrow, spleen, and lymphatic cells. MTP18 Colocalizes and Cofractionates with Mitochondria— To demonstrate specificity of the anti-MTP18 polyclonal antibody we performed immunoblots with lysates derived from HeLa cells transiently transfected with different MTP18 expression constructs. Signals were observed with N-terminal HA-tagged, C-terminal Myc-tagged, and GFP fusion protein constructs indicating the specificity of the anti-MTP18 serum (Fig. 3A). More importantly endogenous MTP18 protein was also detected in whole cell lysates of HeLa cells with the anti-MTP18 antibody. The apparent size of 18 kDa matches the predicted molecular mass deduced from the MTP18 primary sequence (Fig. 3A). To analyze the subcellular localization of MTP18 we performed immunoblot experiments on fractionated cell lysates (Fig. 3B). Fractionation of the cell lysates in cytoplasmic, nuclear, and mitochondrial subfractions revealed that MTP18 cofractionates with the mitochondrial protein Hsp60 (Fig. 3B, lane 4). To demonstrate the quality of the subcellular fractionation we used the catalytic subunit of PI 3-kinase p110α and the transcription factor ETS-1 as cytoplasmic (lane 2) and nuclear markers (lane 3), respectively. To confirm the mitochondrial localization of MTP18 in cells we performed confocal microscopy using the anti-MTP18 antiserum on HeLa cells transfected with MTP18 expression vectors (Fig. 3C, left panel). Costaining with MitoTracker dye revealed a colocalization of mitochondria and MTP18 in transfected HeLa cells (see merged pictures, Fig. 3C, right panel). Notably, N-terminal-tagged GFP-MTP18 fusion protein (Fig. 3C) as well as N-terminally truncated MTP18Δ1–30 (data not shown) did localize to mitochondria suggesting that MTP18 lacks a mitochondrial presequence targeting signal. Taken together, theses data indicate an intracellular localization of MTP18 to mitochondria in mammalian cells. Protein Expression of MTP18 Is Dependent on PI 3-Kinase Signaling—Reduction of mRNA level after LY294002 treatment in PTEN-deficient PC-3 cells suggests that the expression of MTP18 is dependent on PI 3-kinase signaling. To demonstrate the PI 3-kinase-dependent protein expression we analyzed MTP18 protein level in PTEN-positive differentiation-competent HaCaT keratinocytes in response to serum. Whole lysates of HaCaT cells grown in the absence or after stimulation with serum were analyzed by immunoblot (Fig. 4A). Serum depletion led to time-dependent reduction of MTP18, whereas the mitochondrial protein Hsp60 and the PI 3-kinase subunits p110α, p85, and Akt1/2 were not affected. (Fig. 4A, compare lane 1 with lanes 2–5). Phosphorylation of Akt was also decreased in a time-dependent manner after serum deprivation. Interestingly, restimulation by adding fresh serum led to an increase of MTP18 in a time-dependent manner (Fig. 4A, lanes 6–9). Stimulation of Akt phosphorylation by serum addition was more rapid, which is consistent with the idea that Akt kinase is a direct downstream effector of PI 3-kinase. To confirm the dependence of MTP18 protein expression on PI 3-kinase signaling, we employed the PI 3-kinase inhibitor LY294002 in experiments with PTEN-deficient PC-3 cells. Immunoblot analysis of PC-3 cell lysates prepared at different time points after LY294002 (10 μm) treatment showed a rapid decrease in Akt phosphorylation and a more delayed reduction of MTP18 protein expression (Fig. 4B). To verify the results obtained with the small molecule PI 3-kinase inhibitor LY294002, we used our antisense gene silencing tools, GBs (third generation antisense molecules (17Sternberger M. Schmiedeknecht A. Kretschmer A. Gebhardt F. Leenders F. Czauderna F. Von Carlowitz I. Engle M. Giese K. Beigelman L. Klippel A. Antisense Nucleic Acid Drug Dev. 2002; 12: 131-143Google Scholar)) to inhibit gene expression of different members of the PI 3-kinase pathway. We have shown previously that reduction of the PI 3-kinase subunit p110β, but not p110α or Akt1/2, leads to an inhibition of invasive growth of HeLa cells on matrigel (15Czauderna F. Fechtner M. Aygun H. Arnold W. Klippel A. Giese K. Kaufmann J. Nucleic Acids Res. 2003; 31: 670-682Google Scholar, 25Czauderna F. Santel A. Hinz M. Fechtner M. Durieux B. Fisch G. Leenders F. Arnold W. Giese K. Klippel A. Kaufmann J. Nucleic Acids Res. 2003; 31: e127Google Scholar). Similarly, GB-mediated knockdown of p110α, Akt1, and Akt2 did not reduce the growth of PC-3 cells on extracellular matrix, whereas inhibition of p110β decreased growth dramatically (Fig. 4C). We have verified the knockdown of the individual proteins and the status of Akt phosphorylation by immunoblot analysis in comparison with corresponding MM controls (Fig. 4D). With respect to MTP18 expression only reduction of the catalytic subunit p110β decreased the MTP18 protein level (Fig. 4D, lane 3). Neither GB-mediated p110α nor Akt1/2 inhibition reduced the MTP18 protein level. These data suggest that MTP18 protein expression as well as PC-3 cell growth on the extracellular matrix is dependent exclusively on the catalytic subunit p110β but not p110α or Akt1/2 (Fig. 4D, lanes 1, 5, 7, and 9). From these data we conclude that the expression of this novel mitochondrial protein is dependent on PI 3-kinase but not necessarily dependent on signaling via Akt. Inhibition of MTP18 Protein Expression Leads to Reduced Cell Growth—Next, we wanted to analyze the loss-of-function phenotype of MTP18. We performed a screen to identify antisense molecules (GBs), which are potent in reducing MTP18 protein expression. Fig. 5A shows the result of a transfection experiment in PC-3 cells with two different GBs or their corresponding mismatch controls (four mismatches were introduced, see “Experimental Procedures.” Transfection of both GBs reduced the MTP18 protein level significantly at a 15 nm concentration 48 h post-transfection (Fig. 5A, lanes 6 and 13). We used the mitochondrial protein Hsp60 as a control for assaying equal levels of mitochondrial proteins in the whole cell lysates. In parallel we analyzed whether the GB-transfected PC-3 cells showed impaired growth on extracellular matrix or in plastic two-dimensional cultures, similiar to PI 3-kinase catalytic subunit p110β (Fig. 4C). Independently of the growth conditions, we observed growth inhibition of PC-3 cells with reduced MTP18 protein level (Fig. 5B). These data suggest that MTP18 is an essential protein in PC-3 cells. The GB-mediated inhibition of cell proliferation was also demonstrated more quantitatively and over a longer time period in a WST-1 metabolic activity assay (Fig. 5C). Again, the transfection of MTP18-specific GBs, but not of the corresponding mismatches, led to a dramatically reduced WST signal in this assay (Fig. 5C). The GB-mediated inhibition was even more pronounced than the LY294002 treatment (10 μm in Me2SO). To analyze whether the reduced cell growth was due to an inhibition of cell cycle progression or an increase in apoptosis, we performed a FACS analysis. Interestingly, we did not observe a dramatic difference in cell cycle progression in GB- or mismatch-transfected PC-3 cells (Fig. 5D). However, with both MTP18-specific GBs a significant increase in the subG1 DNA fraction was observed indicating an increase in nuclei with reduced DNA content. This result suggests that a reduction of MTP18 protein in PC-3 cells leads to an increase in DNA fragmentation. Knockdown of MTP18 in HaCaT Cells—Since PC-3 cells represent a highly transformed cell line we studied MTP18 loss-of-function phenotypes in normal human keratinocytes (HaCaT) (18Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Google Scholar). Analogous to the growth inhibition of PC-3 cells knockdown of MTP18 in HaCaT cells led to a dramatic reduction in cell proliferation (Fig. 6A). In this cell system cell growth was not only stopped, but the cell number decreased over time after GB treatment. It is important to note that the mismatch containing GBs did not show an anti-proliferative effect demonstrating that the transfection condition are not affecting normal cell proliferation. To verify the specific MTP18 protein knockdown, we performed an immunoblot analysis with lysates from GB-transfected cells. MTP18 protein knockdown was already observed 15 h post-transfection with both GBs (Fig. 6C, upper panel). No reduction in protein expression was observed with the mismatch controls. The mitochondrial protein Hsp60 or the cytoplasmic p110α served as loading control. As mentioned above, we observed a decrease in cell number in transfection experiments with MTP18-specific GB. FACS analysis with the transfected HaCaT cells showed similar results as obtained with PC-3 cells observing an increase in the subG1 fraction (Fig. 6B). The increase of cells in the subG1 fraction points again toward an apoptotic DNA fragmentation in cells with reduced MTP18 protein level. It is well established that cytochrome c release from mitochondria into cytoplasm activates caspases, and in turn activated caspase-3 cleaves the death substrate PARP (26Lazebnik Y.A. Kaufmann S.H. Desnoyers S. Poirier G.G. Earnshaw W.C. Nature. 1994; 371: 346-347Google Scholar). To address the question whether the inhibition of MTP18 protein can stimulate apoptosis, we performed immunoblot analysis with cleaved PARP and cytochrome c-specific antibodies on fractionated lysates derived from GB transfected cells. We observed with both GBs an increase in cleaved PARP signal in the cytoplasmic fraction (Fig. 6C, lanes 4 and 6 and 12 and 14). As a positive control we used lysates from UV-treated HaCaT cells (Fig. 6C, lanes 8 and 16). Knockdown of MTP18 as well as UV treatment led also to the release of cytochrome c from the mitochondrial fraction into the cytoplasm demonstrating the onset of apoptosis. This result suggests that loss-offunction of MTP18 protein induces apoptosis similar to UV treatment in HaCaT cells. Reduction of MTP18 Results in Increased Response to Apoptotic Stimuli—In the next step we wanted to analyze whether UV- or TNFα-induced apoptosis is altered in HaCaT cells with reduced MTP18 protein level. HaCaT cells were transfected with MTP18-specific GB or the corresponding mismatch-containing molecules. 24 h later the transfected and untreated cells (UT) were exposed to two different doses of UV (Fig. 7A) or treated with two different concentrations of TNFα (Fig. 7B). The activation of apoptosis was measured by immunoblot analysis detecting cleaved PARP. In cells with GB1- and GB2-mediated MTP18 protein reduction we observed with both UV doses and both TNFα concentrations an increase in cleaved PARP signal when compared with mismatch-, UV-, or even TNFα-only-treated cells (Fig. 7, lanes 5, 8, 14, and 17). We concluded from these data that HaCaT cells with reduced MTP18 protein levels are more susceptible to UV- or TNFα-induced apoptosis. Changes in MTP18 Expression Affect Mitochondrial Morphology—Loss of MTP18 function caused cytochrome c release and apoptosis in HaCaT cell pools. To investigate loss-of-function effect of this novel mitochondrial protein in its cellular context, we were interested in studying this effect on single cell level. COS-7 cells were transfected with two MTP18-specific GBs or the corresponding MM controls. Cells were analyzed 24 h after transfection by immunoblotting to confirm specific reduction of MTP18 protein in GB-transfected cells (Fig. 8A). The transfected COS-7 cells were analyzed by immunofluorescence using anti-cytochrome c antibody to assess mitochondrial shape and cytochrome c distribution. Cells transfected with MTP18-specific GBs, but not with their corresponding MM controls, showed release