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(American Journal of Pathology. 2004;165:553-564.)
© 2004 American Society for Investigative Pathology

Expression of Histone Deacetylase 8, a Class I Histone Deacetylase, Is Restricted to Cells Showing Smooth Muscle Differentiation in Normal Human Tissues

David Waltregny*{dagger}, Laurence de Leval{ddagger}, Wendy Glénisson*, Siv Ly Tran*, Brian J. North§, Akeila Bellahcène*, Ulrich Weidle, Eric Verdin§ and Vincent Castronovo*

From the Metastasis Research Laboratory and Center of Experimental Cancer Research* and the Departments of Urology{dagger} and Pathology,{ddagger} University of Liège, Liège, Belgium; the Gladstone Institutes of Virology and Immunology,§ University of California, San Francisco, California; and Roche Diagnostics, Penzberg, Germany


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Histone deacetylases (HDACs) were originally identified as nuclear enzymes involved in gene transcription regulation. Until recently, it was thought that their activity was restricted within the nucleus, with histones as unique substrates. The demonstration that specific HDACs deacetylate nonhistone proteins, such as p53 and {alpha}-tubulin, broadened the field of activity of these enzymes. HDAC8, a class I HDAC, is considered to be ubiquitously expressed, as suggested by results of Northern blots performed on tissue RNA extracts, and transfection experiments using various cell lines have indicated that this enzyme may display a prominent nuclear localization. Using immunohistochemistry, we unexpectedly found that, in normal human tissues, HDAC8 is exclusively expressed by cells showing smooth muscle differentiation, including visceral and vascular smooth muscle cells, myoepithelial cells, and myofibroblasts, and is mainly detected in their cytosol. These findings were confirmed in vitro by nucleo-cytoplasmic fractionation and immunoblot experiments performed on human primary smooth muscle cells, and by the cytosolic detection of epitope-tagged HDAC8 overexpressed in fibroblasts. Immunocytochemistry strongly suggested a cytoskeleton-like distribution of the enzyme. Further double-immunofluorescence staining experiments coupled with confocal microscopy analysis showed that epitope-tagged HDAC8 overexpressed in murine fibroblasts formed cytoplasmic stress fiber-like structures that co-localized with the smooth muscle cytoskeleton protein smooth muscle {alpha}-actin. Our works represent the first demonstration of the restricted expression of a class I HDAC to a specific cell type and indicate that HDAC8, besides being a novel marker of smooth muscle differentiation, may play a role in the biology of these contractile cells.

Histone deacetylases (HDACs) represent a large family of enzymes identified as key regulators of nucleosomal histone acetylation, a major event that controls eukaryotic gene transcription.1,2 Most HDACs can function as transcriptional co-repressors and are often present in large multisubunit complexes, such as the sin3 complex.3-6 Currently, at least 18 different members of the HDAC family have been isolated from mammalian cells7 and are classified into three groups: 1) class I members, including HDAC1, HDAC2, HDAC3, HDAC8, and HDAC11, which are homologous to the yeast Rpd3 protein;8-13 2) class II HDACs, including HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, which have similarities to yeast Hda1;14-20 and, 3) NAD-dependent sirtuin (SIRT) proteins, which are homologous to the yeast Sir2 protein.21-23 Although the precise functions of each member of the HDAC and SIRT families are primarily unknown, accumulating evidence suggests that they are involved in important biological activities, such as cell differentiation, proliferation, apoptosis, and senescence.24

Although it has been initially considered that HDACs’ activities are restricted to the nucleus for deacetylation of nucleosomal histones, recent observations have indicated that this is not always the case. Indeed, it appears that some HDACs can deacetylate nonhistone nuclear and cytoplasmic proteins, such as the tumor suppressor p5325-27 and the cytoskeletal protein {alpha}-tubulin.28 Interestingly, the deacetylation of these proteins affects their activity. Based on these observations, it is expected that HDACs might exert, within the cell, much broader biological activities than the exclusive control of gene transcription.

Despite the unanimous recognition that HDACs represent a family of key enzymes with paramount activities for the cell, information regarding the exact role of each individual HDAC has remained scarce. One approach to explore the function(s) of specific HDACs is to examine their profile of expression in specific tissues. We have successfully used such a strategy to recently demonstrate that HDAC7 acts as a regulator of Nur77 and apoptosis in developing thymocytes.29

Class I HDACs have ~350 to 500 amino acids and their transcript expression is considered to be ubiquitous.30 Class II HDACs are much larger proteins with ~1000 amino acids; their mRNA distribution is more restricted and they are implicated in the development and differentiation of cardiac and skeletal striated muscle.31 Class II enzymes can shuttle in and out of the nucleus on certain cellular signals.32 Among class I members, HDAC1 and HDAC2 are localized exclusively in the cell nucleus30 whereas HDAC3 can be detected in the nuclear and cytoplasm compartments.33

Database searches for expressed sequence tags showing high similarity with class I HDACs have led to the cloning of the cDNA for human HDAC8, the fourth identified class I HDAC.10,11,13 This enzyme encodes 377 amino acid residues and is evolutionary most similar to HDAC3 with 34% overall identity. Although Northern blot analyses have revealed that HDAC8 mRNA expression is ubiquitous,10,11,13 but distinct from that of HDAC1 and HDAC3,10 no data have been available on the distribution of the enzyme in human tissues.

As a result of a screening of HDAC expression profiles in human prostate tissues, we have observed that HDAC8, rather than being detected in all cell types, is exclusively expressed by some prostate stromal cells as well as by cells present in vascular walls. We have extended our investigation to most human tissues and herein demonstrate that HDAC8 is exclusively expressed by normal human cells showing smooth muscle differentiation in vivo, including vascular and visceral smooth muscle cells, myoepithelial cells, and myofibroblasts. Unexpectedly, the enzyme is predominantly cytosolic, both in human tissues and in in vitro grown human vascular smooth muscle cells where it displays a cytoskeleton-like pattern of expression. We provide evidence that HDAC8 may co-localize with the smooth muscle cytoskeleton protein smooth muscle {alpha}-actin ({alpha}-SMA). Altogether, our results unveil HDAC8 as a novel biological marker of the smooth muscle phenotype and suggest a specific involvement for this HDAC in smooth muscle differentiation.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines, Tissue Culture, and Reagents

NIH-3T3 mouse embryonic fibroblast and HeLa human cervix epithelial cell lines were purchased from the American Type Culture Collection (Manassas, VA). Primary human skin fibroblasts were established by outgrowth of normal human skin biopsies as detailed elsewhere.34,35 Primary human smooth muscle cells (HSMCs) were harvested from human umbilical cord veins, essentially as previously described,35,36 after removal of the endothelial cell layer.37 Briefly, umbilical cord veins were cannulated and flushed with 50 ml of RPMI 1640 culture medium (Invitrogen, Merelbeke, Belgium) to remove blood, and allowed to drain. The vein was then filled with 1 mg/ml of collagenase A (Roche, Mannheim, Germany) in RPMI 1640 and incubated at 37°C in a bath containing sterile Dulbecco’s phosphate-buffered saline (PBS) without calcium, magnesium, and sodium bicarbonate for 10 minutes. Endothelial cells were removed by thoroughly flushing with 50 ml of RPMI 1640. The vein was further rinsed with 50 ml of RPMI 1640 before reintroducing collagenase A in its lumen. After incubation at 37°C for 20 minutes, smooth muscle cells were harvested by thoroughly flushing with 50 ml of RPMI 1640, centrifuged, resuspended in culture medium, and seeded onto Petri dishes. All cells were grown and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% decomplemented (heat-inactivated) fetal bovine serum (Biowhittaker, Walkersville, MD), 50 U/ml penicillin, 50 µg/ml streptomycin, 0.1% fungizone, and 2 mmol/L L-glutamine at 37°C in a humidified 95% air/5% CO2 atmosphere. Primary human cells were used between passages 7 and 13. In vitro grown HSMCs displayed a typical smooth muscle cell morphology and immunoblot experiments enabled to check that, at passages 7 through 13, HSMCs retained {alpha}-SMA and smooth muscle myosin heavy chain (SMMHC) expression (data not show). All tissue culture reagents were obtained from Invitrogen unless otherwise specified.

Tissues

Formalin-fixed paraffin-embedded normal human tissue samples were obtained from the Department of Pathology at the University Hospital of Liège, Belgium. The organs from which the tissues were sampled are listed in Table 1 .


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  		Table 1. Localization of HDAC8 Expression in Various Normal Human Tissues and Organs

 
Cloning of HDAC8, Transfections, and Immunofluorescence

Total RNA was extracted from HSMCs using the RNeasy mini kit (Qiagen, Inc., Valencia, CA), according to the manufacturer’s protocol. To obtain the full-length human HDAC8 cDNA, a reverse transcriptase-polymerase chain reaction amplification of HSMC total RNA was set up with the use of the Pfu DNA polymerase (Promega, Leiden, The Netherlands) and the following primers: 5'-CACCATGGAGGAGCCGGAGGAA-3' (GenBank NM_018486 bases 43 to 60) and 5'-GACCACATGCTTCAGATTCCCTT-3' (GenBank NM_018486 bases 1173 to 1149).pcDNA3.1D/HDAC8/V5-His was constructed by directional cloning of the HDAC8 coding sequence upstream of and in-frame with the carboxy-terminal V5 epitope and hexahistidine sequence into pcDNA3.1D/V5-His-Topo (Invitrogen). Mock transfection with transfecting reagent alone and transfection with pcDNA3.1D/lacZ/V5-His plasmid (Invitrogen) served as controls. The HDAC8 construct was checked by DNA sequencing of the insert and multiple cloning sites. Human HDAC3, HDAC6, and HDAC8 cDNAs were also subcloned to generate C-terminal FLAG-tagged fusions into a FLAG vector [a derivative of the pcDNA3.1(+) vector backbone (Invitrogen)], as previously described.38,39

NIH-3T3 cells grown on coverslips in 35-mm dishes were transfected at a density of 3 x 104 cells/cm2 with 2 µg of pcDNA3.1D/HDAC8/V5-His or pcDNA3.1D/lacZ/V5-His plasmid and 6 µl of FUGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN), according to the manufacturer’s directions. Cells were cultured for 24 hours, washed twice with PBS (10 mmol/L sodium phosphate and 0.9% NaCl, pH 7.4), and either fixed with 2% formaldehyde for 15 minutes or subjected to lysis for total protein extraction. Detection of V5-tagged proteins was performed by Western blot analysis, as detailed below. Indirect immunofluorescence was used to show the subcellular localization of V5-tagged HDAC8. Briefly, after fixation and two washes in PBS, the endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30 minutes. After washes in distilled water for 5 minutes and in PBS for 20 minutes, cells were permeabilized with 0.2% Triton X-100 (Sigma Chemical Co., St. Louis, MO) for 5 minutes on ice. The slides were then incubated with 3% normal horse serum (Vector Laboratories Inc., Burlingame, CA) in PBS for 30 minutes to block the nonspecific serum-binding sites. Anti-V5 antibody (Invitrogen) at a dilution of 1:500 was applied and incubated for 1 hour, followed by incubation with a biotinylated horse anti-mouse IgG antibody and the avidin-biotin-peroxidase complex ABC (Vectastain Elite immunoperoxidase kit; Vector Laboratories, Inc.). After each incubation, the slides were washed three times with 1% normal horse serum in PBS for 5 minutes. Peroxidase activity was developed with a solution containing fluorescein isothiocyanate-conjugated tyramine in amplification diluent (NEN, Boston, MA). After three washes in PBS for 10 minutes, the cells were counterstained with 4',6'-diamidino-2'-phenylindole dichloride (Roche Diagnostics) and the coverslips were mounted with anti-fading fluorescent mounting medium (DAKO, Carpinteria, CA) for microscopic examination. Color photomicrographs of the slides were taken with an Axioplan fluorescence microscope (Zeiss) equipped with appropriate filter sets.

For double-immunofluorescence staining experiments, NIH-3T3 cells grown on coverslips were transfected with pcDNA3.1(+)/HDAC3/FLAG, pcDNA3.1(+)/HDAC6/FLAG, or pcDNA3.1(+)/HDAC8/FLAG and FUGENE 6 transfection reagent. NIH-3T3 cells transfected with pcDNA3.1(+)/FLAG alone served as negative control. Transfected cells were processed for immunofluorescence microscopy 48 hours after transfection. Cells on coverslips were washed twice in PBS for 10 minutes, fixed in 4% paraformaldehyde for 10 minutes, followed by permeabilization in 0.5% Triton X-100 in PBS for 10 minutes. After three washes for 10 minutes each in PBS, cells were incubated for 10 minutes in 10% bovine serum albumin in PBS and then incubated for 1 hour with anti-{alpha}-SMA or anti-FLAG antibodies or together, each diluted 1:500 in PBS + 0.1% Tween-20. Cells were washed three times for 10 minutes in PBS containing 0.1% Tween-20, followed by incubation for 1 hour with donkey anti-mouse IgG Cy2-conjugated or donkey anti-rabbit IgG Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or together, each diluted 1:500 in PBS + 0.1% Tween-20. Cells were then washed three times for 10 minutes each in PBS and once briefly in ddH2O, and mounted on slides with Gel Mount (Biomeda Corp., Foster City, CA). Confocal images were acquired by laser-scanning confocal microscopy with an Olympus BX60 microscope equipped with a Radiance 2000 confocal setup (Bio-Rad, Hercules, CA).

Antibodies

Anti-HDAC8 antibody (N-20) was raised against an epitope mapping at the N-terminus of human HDAC8 (Santa Cruz Biotechnology, Santa Cruz, CA). Polyclonal rabbit anti-HDAC1 (no. 2062), anti-HDAC3 (no. 2632), and anti-HDAC5 (no. 2082) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-{alpha}-tubulin (clone B512), anti-{alpha}-SMA (clone 1A4), and anti-SMMHC (M-7786) monoclonal antibodies as well as anti-FLAG (F-7425) rabbit polyclonal antibody were from Sigma (Bornem, Belgium).

Protein Extraction

HDAC8 protein expression was examined in primary HSMCs and fibroblasts and in HeLa epithelial cells. After rinses in PBS (PBS Dulbecco’s without calcium, magnesium, and sodium bicarbonate), in vitro grown subconfluent cells were scrapped and pelleted by centrifugation at 300 x g for 10 minutes. Total protein lysates were obtained by incubating cell pellets with protein lysis buffer containing 0.1% Triton X-100, 50 mmol/L Tris (pH 7.5), 250 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, 1 mmol/L phenylmethyl sulfonyl fluoride, and Complete protease inhibitor cocktail (Roche). Protein lysates were placed in ice for 30 minutes, vortexed every 10 minutes, and then cleared by centrifugation at 12,000 x g for 20 minutes at 4°C. The supernatants were retrieved and frozen at –80°C until use in immunoblot assays. The protein concentration was measured using a bicinchoninic acid determination kit (Pierce Chemical Co., Rockford, IL).

Immunoblotting

Equal amounts of protein lysates were resolved by size on 10% Bis-Tris-polyacrylamide gels (Invitrogen) and transferred onto polyvinylidene difluoride membranes (Roche Diagnostics, Mannheim, Germany), which were stained with Ponceau S (Sigma Chemical Company) to examine the equal protein sample loading and transferring (data not shown). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (20 mmol/L Tris base, pH 7.6, 150 mmol/L NaCl) containing 0.1% Tween-20 (TBS-T), and probed with the following primary antibodies: anti-HDAC1, anti-HDAC3, anti-HDAC8, and anti-{alpha}-tubulin. After washing in TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories) and developed using an enhanced chemiluminescence detection system (ECL detection kit; Amersham Corp., Arlington Heights, IL), according to the instructions of the manufacturer. Membranes were exposed to Kodak X-Omat AR films (Eastman-Kodak, Rochester, NY).

Nuclear and Cytoplasmic Fractionation

Approximately 107 cells were collected and washed twice in ice-cold PBS. The cell pellet was resuspended in 1 ml of washing buffer (10 mmol/L Hepes, pH 7.9, 20 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, and protease inhibitors), microcentrifuged at 100 x g for 2 minutes at 4°C, and then lysed in 500 µl of buffer A (10 mmol/L Hepes, pH 7.9, 10 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.2% Nonidet P-40, 1 mmol/L dithiothreitol, and protease inhibitors). After incubation on ice for 30 seconds, nuclei were pelleted by microcentrifugation at 3500 x g for 5 minutes at 4°C and supernatant was collected as the cytoplasmic fraction. The nuclei pellet was washed three times in 500 µl of washing buffer and then resuspended in 1 vol of buffer B (20 mmol/L Hepes, pH 7.9, 630 mmol/L NaCl, 1.5 mmol/L MgCl2, 25% glycerol, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, and protease inhibitors). The suspension was mixed gently by rocking for 45 minutes at 4°C and then centrifuged at 14,000 x g for 30 minutes at 4°C. Supernatant was collected as the nuclear fraction. The amount of HDAC1, HDAC3, HDAC8, and {alpha}-tubulin in the fractionated nuclear and cytoplasmic cell extracts was analyzed by immunoblotting, as detailed above.

Immunoperoxidase

Detection of HDAC8 protein in human tissues and cells was performed with the use of an immunoperoxidase technique and the ABC Vectastain Elite kit (Vector Laboratories, Inc.) according to the supplier’s directions with some modifications. Primary human skin fibroblasts, murine NIH-3T3 fibroblasts, and HSMCs were seeded onto poly-L-lysine-coated glass slides, grown to 70 to 80% confluence, washed with PBS, and then fixed with freshly prepared 2% formaldehyde for 15 minutes. Five-µm formalin-fixed paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in graded alcohols. After blocking of the endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol for 30 minutes, the sections were heated in a water-bath at 95°C in citrate buffer, allowed to cool down, and then incubated with 1% normal horse serum ({alpha}-SMA and SMMHC), 1% normal goat serum (HDAC5), or 1% normal swine serum (HDAC8) in PBS for 30 minutes. For anti-SMMHC immunostaining, an additional trypsinization step was performed as previously described,40 before heating the sections for antigen retrieval; tissue sections were incubated with 0.125 mg/ml trypsin (Life Technologies, Inc., Grand Island, NY) in PBS for 20 minutes at 37°C and then washed with PBS for 20 minutes. Mouse anti-{alpha}-SMA antibody at a dilution of 1:400, mouse anti-SMMHC antibody at a dilution of 1:2500, rabbit anti-HDAC5 antibody at a dilution of 1:500 or goat anti-HDAC8 antibody at a dilution of 1:200 was incubated overnight at 4°C, followed by biotinylated horse anti-mouse, goat anti-rabbit, or swine anti-goat IgG antibody and the avidin-biotin-peroxidase complex. Washes were performed three times with PBS after each incubation step. Peroxidase activity was developed by a solution of (Vel, Leuven, Belgium) dissolved in PBS and 0.03% H2O2. The 3–3' diaminobenzidine tetrahydrochloride solution was filtered and applied to the sections for 4 minutes. Finally, Carazzi’s hematoxylin was used to counterstain the slides that were then dehydrated and mounted. Control experiments included omission of the first antibody and preincubation of anti-HDAC8 antibody with a 50-molar excess of the corresponding peptide before the antibody’s use in the immunoperoxidase assay. Masson’s trichrome staining was performed on paraffin-embedded tissues as previously described41 to delineate collagen and smooth muscle fibers. Photomicrographs of the slides were taken with a Zeiss microscope.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
HDAC8 Expression in Normal Human Tissues Is Restricted to Cells Showing Smooth Muscle Differentiation

Immunohistochemistry was performed with the use of a specific anti-HDAC8 antibody to assess HDAC8 expression in a large number of normal human tissue types and organs. Figures 1 and 2 show representative photomicrographs of anti-HDAC8 immunoreactivity. Masson’s trichrome staining was used to distinguish collagen and smooth muscle fibers present in the wall of small arteries (Figure 1A) . As shown in Figure 1C , anti-HDAC8 immunoreactivity was detected only in the smooth muscle cells of the vascular walls. Control immunohistochemical experiments in which the anti-HDAC8 antibody had been preincubated with a molar excess of the corresponding peptide completely abolished the labeling (Figure 1D) . Similarly, no specific staining was observed when the primary antibody was replaced with PBS in the immunoperoxidase procedure (Figure 1B) .



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  		Figure 1. A: Masson’s trichrome staining of arterial walls present in the fat tissue surrounding a human prostate gland. Smooth muscle fibers appeared violet and collagen fibers were stained in green. HDAC8 expression was analyzed in serial tissue sections of the same arterial walls as in A using an immunoperoxidase technique, as described in Materials and Methods. B: Control immunohistochemical experiment in which the anti-HDAC8 antibody had been replaced with PBS. C: Incubation of the section with anti-HDAC8 antibody yielded a specific intense immunoreactivity in smooth muscle cells from the arterial walls. D: Control experiment in which anti-HDAC8 antibody had been preincubated with a molar excess of the corresponding peptide. E: Immunohistochemical detection of HDAC5 in the nucleus of human cardiomyocytes. F: Detection of HDAC8 by immunohistochemistry in myocardium. Neither cardiomyocytes nor endothelial cells from small arteries exhibited a detectable level of HDAC8 expression whereas vascular smooth muscle cells harbored strong anti-HDAC8 immunoreactivity. Note a detectable level of HDAC8 expression in a small capillary wall. G: Strong diffuse cytoplasmic anti-HDAC8 immunoreactivity in smooth muscle cells from a small artery wall in a liver portal space. Note the absence of specific reactivity in an adjacent nerve. H: Strong diffuse anti-HDAC8 reactivity in the cytosol of smooth muscle cells from colon muscularis mucosae and from arterial and capillary walls. Note detectable HDAC8 expression in scattered cells with myofibroblastic features (intestinal subepithelial myofibroblasts) located in the lamina propria. Tissue sections were counterstained with hematoxylin. Original magnifications: x100 (A–D); x200 (E, F); x400 (G, H).

 
Anti-HDAC8 immunoreactivity, characterized by intense diffuse cytoplasmic staining, was exclusively detected in vivo in human cells showing smooth muscle differentiation, in extenso visceral and vascular smooth muscle cells, myoepithelial cells, and myofibroblasts. Table 1 details the distribution of HDAC8 expression in the various human tissues analyzed. As shown in Figures 1 and 2 , HDAC8 expression was detected in vascular smooth muscle cells, including large vessel (arteries and veins) and microvessel (arterioles, venules, and capillaries) smooth muscle cells. Anti-HDAC8 immunoreactivity was also found in smooth muscle cells from all smooth muscle-containing organs tested, such as intestine (Figure 1H) , lung (Figure 2H) , fallopian tubes (Figure 2K) , and bladder (Figure 2L) .



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  		Figure 2. Immunodetection of HDAC8 in various human organs and tissues. Formalin-fixed, paraffin-embedded human tissue sections were subjected to detection of HDAC8 by immunoperoxidase, as described in Materials and Methods. HDAC8 expression was investigated in thyroid (A), brain (B), thymus (C), testis (D), pancreas (E), liver (hepatocytes) (F), skeletal muscle (G), lung (H), breast (I), uterine cervix (J), fallopian tube (K), and bladder wall (L). Sections were counterstained with hematoxylin. Original magnifications: x100 (C, H); x200 (J, K, L); x400 (A, B, E, G); x630 (D, F, I).

 
HDAC8 expression was also searched in myoepithelium-bearing glands, including mammary gland alveoli and ducts, respiratory tract mucous glands, salivary glands, and skin eccrine glands. HDAC8 was detected in myoepithelial cells from all these glandular structures (Figures 2I and 3 , and data not shown). To assess the possible co-localization of HDAC8 with {alpha}-SMA and SMMHC, two well-known smooth muscle-specific cytoskeleton proteins,40,42,43 serial sections from normal human breast, trachea, salivary glands, and skin tissues were subjected to immunohistochemistry using specific anti-HDAC8, anti-{alpha}-SMA, and anti-SMMHC antibodies. Our results showed that myoepithelial cells from mammary acini/ducts, tracheal mucous glands, salivary glands, and skin eccrine glands co-expressed the three proteins (Figure 3 and data not shown).



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  		Figure 3. Immunodetection of HDAC8, {alpha}-SMA, and SMMHC in myoepithelial cells from normal human tracheal mucous glands and mammary acini/ducts. Serial sections of these tissues were subjected to detection of HDAC8, {alpha}-SMA, and SMMHC by immunoperoxidase, as described in Materials and Methods. Original magnifications, x400.

 
HDAC8 expression was also detected in various myofibroblasts-containing tissues. Figures 1H and 2D represent photomicrographs of intestine and testis tissue sections, respectively, which were immunostained with anti-HDAC8 antibody. As observed in Figure 1H , HDAC8 expression was found in smooth muscle cells from vascular walls, in smooth muscle cells from muscularis mucosae, as well as in intestinal subepithelial myofibroblasts present in the lamina propria and extending from the muscularis mucosae to the subepithelial aspect of the surface epithelium. In testis, HDAC8 expression was observed in a thin layer of myofibroblasts (peritubular myoid cells) located around seminiferous tubules. Myofibroblastic cells from several, but not all, other myofibroblasts-containing normal human tissues analyzed also exhibited HDAC8 expression. Indeed, in addition to its detection in testis peritubular myoid cells and intestine subepithelial myofibroblasts, HDAC8 was also expressed by lung alveolar septae myofibroblasts, prostate stromal cells, reticular cells of the spleen, and external theca cells of the ovary (Figure 4 and data not shown). Interestingly, like myoepithelial cells, all these myofibroblastic cells co-expressed HDAC8, {alpha}-SMA, and SMMHC (Figure 4 and data not shown). No specific anti-HDAC8 immunoreactivity was detected in some other types of myofibroblastic cells, such as reticular cells of the thymus (Figure 2C) , stromal cells of the breast (Figures 2I and 3) , periacinar stellate cells of the pancreas (Figure 2E) , perisinusoidal stellate (Ito) cells of the liver (Figure 2F) , and mesangial cells of the kidney (data not shown).



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  		Figure 4. Immunodetection of HDAC8, {alpha}-SMA, and SMMHC in myofibroblastic cells from normal human intestine, lung, and spleen. Intestinal subepithelial myofibroblasts, lung alveolar septae myofibroblasts, and spleen reticular cells co-expressed HDAC8, {alpha}-SMA, and SMMHC. Serial sections of these tissues were subjected to detection of HDAC8, {alpha}-SMA, and SMMHC by immunoperoxidase, as described in Materials and Methods. v = vessel; arrows indicate myofibroblastic cells. Original magnifications, x400.

 
HDAC8 protein was not detected in nonsmooth muscle cells. In particular, striated muscle (Figures 1F and 2G) , neuronal (Figures 1G and 2B) , endothelial (Figures 1F, 2E, and 2G) , and epithelial (Figures 1H, 2A, 2E, 2F, 2H, 2I, 2J, 2K, and 4) cells as well as osteocytes (data not shown), chondrocytes (Figure 2H) , lymphocytes (Figure 2C and 4) , and fibrocytes (Figures 1, 2, and 3) were devoid of anti-HDAC8 immunoreactivity.

HDAC8 expression was also investigated by immunoblot in in vitro grown HSMCs, human skin fibroblasts, and human HeLa cervix epithelial cells. Among the three cell lines, the abundance of HDAC8 was the highest in HSMCs, with lower levels in human fibroblasts (Figure 5A) . We had observed that cervix keratinocytes from normal human tissues did not express detectable levels of HDAC8 (Figure 2J) . Similarly and as previously described,44 in vitro grown HeLa human cervix epithelial cells exhibited no detectable expression of the enzyme (Figure 5A) .



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  		Figure 5. A: Protein lysates from primary human skin fibroblasts, primary HSMCs, and HeLa human cervix epithelial cells were subjected to immunoblot analysis of HDAC8 expression, as described in Materials and Methods. HDAC8 expression was investigated in human primary skin fibroblasts and smooth muscle cells from human umbilical cord vein (HSMC), by immunoperoxidase, as described in Materials and Methods. B: Anti-HDAC8 immunostaining presented a pattern suggestive of a cytoskeletal association in HSMCs. C: Anti-HDAC8 immunoreactivity in HSMCs was abolished by preincubating anti-HDAC8 antibody with a molar excess of the corresponding peptide. D: Primary human skin fibroblasts usually exhibited no detectable level of anti-HDAC8 immunoreactivity. E and F: Detection of HDAC8 in the cytoplasm (E) or nucleus (F) of NIH-3T3 cells. Original magnifications: x630 (B); x400 (C, D); x200 (E, F).

 
HDAC8 Is a Predominantly Cytoplasmic HDAC and Co-Localizes with {alpha}-SMA

In all human tissues in which HDAC8 expression was detectable, the enzyme was present mainly in the cytoplasmic compartment of the enzyme-expressing cells (Figures 1 through 4) . This contrasted with the subcellular localization of HDAC5, a class II HDAC involved in skeletal muscle differentiation. Indeed, HDAC5 expression was observed in the nucleus of cardiac myocytes (Figure 1E) .

Immunocytochemistry experiments also showed the prominent cytoplasm localization of HDAC8 in HSMCs, with a distribution pattern suggestive of a cytoskeletal association (Figure 5B) . To verify the specificity of the anti-HDAC8 immunoreactivity obtained in primary cultures, the antibody was preincubated with a molar excess of the corresponding peptide. The preincubation of the antibody with its peptide produced a complete disappearance of the staining (Figure 5C) . Similarly to fibroblasts in human tissues, cultured fibroblasts usually exhibited no detectable expression of HDAC8 (Figure 5D) . Murine NIH-3T3 fibroblasts displayed both nuclear and cytoplasmic expression of HDAC8 (Figure 5, E and F) . The cytoplasm distribution of HDAC8 in these cells was similar to that observed in HSMCs (Figure 5 , compare B with E).

To further examine the subcellular localization of HDAC8, we performed cell fractionation experiments with the use of primary human fibroblasts, HSMCs, and NIH-3T3 cells. After separation of the cytoplasm and nuclear fractions, HDAC8 was enriched in the cytoplasm fraction, whereas HDAC1 and the cytoskeleton protein {alpha}-tubulin were localized exclusively in the nucleus and in the cytosol, respectively. HDAC3 was mainly localized in the nuclear compartment although it was also detected in the cytosol (Figure 6) .



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  		Figure 6. The amount of HDAC1, HDAC3, HDAC8, and {alpha}-tubulin in the fractionated nuclear and cytosolic extracts from primary human skin fibroblasts, HSMCs, and NIH-3T3 fibroblasts was analyzed by immunoblotting as described in Materials and Methods.

 
Transfection experiments also indicated that HDAC8 could be localized in the cytoplasm. A V5-tagged HDAC8 construct was transiently transfected into NIH-3T3 cells. Forced expression of the tagged enzyme was checked by immunoblot analysis using an anti-V5 antibody (Figure 7A) . Indirect immunofluorescence using the same antibody was used to visualize the cellular localization of transfected HDAC8. As shown in Figure 7B , exogenous HDAC8 was localized both in the cytoplasm and in the nucleus of NIH-3T3 cells.



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  		Figure 7. A: Western blot analysis of transfected recombinant human HDAC8 in NIH-3T3 cells. Total cell extracts from NIH-3T3 cells transiently transfected with pcDNA3.1D/HDAC8/V5-His, as in Materials and Methods, were subjected to immunoblot analysis using an anti-V5 antibody (Invitrogen). Lane designations were as follows: lane 1, NIH-3T3 transiently transfected with pcDNA3.1D/lacZ/V5-His; lane 2, NIH-3T3 transiently transfected with the insert-less vector; lane 3, untransfected NIH-3T3 cells; lane 4, NIH-3T3 transiently transfected with pcDNA3.1D/HDAC8/V5-His. B: Subcellular localization of V5-tagged HDAC8 transfected into NIH-3T3 cells. Left: Representative examples of anti-V5 immunofluorescence, as described in Materials and Methods. Middle and right: 4',6'-diamidino-2'-phenylindole dichloride and merged images, respectively.

 
In cultured HSMCs and NIH-3T3 cells, the distribution of HDAC8 expression was reminiscent of stress fiber-like structures, suggesting a possible association of the enzyme with the smooth muscle cytoskeleton. To investigate whether HDAC8 may associate with smooth muscle cytoskeleton proteins, and in particular with {alpha}-SMA, double-immunofluorescence staining experiments coupled with confocal microscopy analysis were performed with an antibody for {alpha}-SMA and with an anti-FLAG antiserum after transfection of cDNAs encoding either HDAC3, HDAC6, or HDAC8 (C-terminal FLAG-tagged) in NIH-3T3 cells. As shown in Figure 8 , exogenous HDAC8, detected with an anti-FLAG antibody, was present in the nuclear and cytoplasmic compartments. Thus, exogenously expressed HDAC8 displayed an intracellular localization similar to that of endogenously expressed HDAC8 (Figure 5, E and F) . In the cytoplasm, FLAG-tagged HDAC8 displayed a stress fiber-like distribution of expression, and more importantly, co-localized with {alpha}-SMA, mainly as filamentous structures (Figure 8) . No co-localization was found between {alpha}-SMA and either FLAG-tagged HDAC3, which was almost exclusively nuclear, or FLAG-tagged HDAC6, which is known to associate with {alpha}-tubulin (Figure 8) .28,39,45



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  		Figure 8. Double-immunofluorescence staining coupled with confocal microscopy analysis, using anti-{alpha}-SMA and anti-FLAG antibodies after transfection of cDNAs encoding either HDAC3, HDAC6, or HDAC8 (C-terminal FLAG-tagged) in NIH-3T3 cells. NIH-3T3 cells grown on coverslips were transfected with pcDNA3.1(+)/HDAC3/FLAG, pcDNA3.1(+)/HDAC6/FLAG, or pcDNA3.1(+)/HDAC8/FLAG. NIH-3T3 cells transfected with pcDNA3.1(+)/FLAG alone served as negative control. Transfected cells were processed for immunofluorescence microscopy 48 hours after transfection and analyzed using confocal microscopy as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Initial experiments performed on total tissue extracts have indicated that HDAC8 may be ubiquitously expressed in human tissues and organs,10,11,13 with the corollary that the enzyme would exert its deacetylase activity necessary for each cell type. The data of our current study cancel out these assumptions because, as demonstrated herein, HDAC8 protein expression is restricted to a specific human cell type that comprises various cells sharing a common phenotype: smooth muscle cell differentiation. This specialized cell type includes visceral and vascular smooth muscle cells as well as myoepithelial cells and myofibroblasts. Our findings demonstrate the limitations of techniques applied to total tissue or organ extracts for the search of the expression profiles of a specific protein both at the mRNA or protein levels. Immunohistochemistry probably remains the only definitive strategy to elucidate the precise pattern of expression of a specific molecule in an animal body. This technique has enabled us to demonstrate that, rather than being a protein expressed in a ubiquitous manner in all human tissues, HDAC8 is a potential novel smooth muscle cell differentiation marker. Such a statement is supported by the observation that primary HSMCs exhibit substantially higher amounts of HDAC8 than primary human fibroblasts and HeLa cervix epithelial cells.

It has been known for more than 15 years that, similarly to vascular and visceral smooth muscle cells, myoepithelial cells express the {alpha}-SMA isoform and SMMHC, two major proteins of the smooth muscle actomyosin complex.40,43,46,47 In this study, we have demonstrated that myoepithelial cells from all myoepithelium-bearing glands analyzed, including mammary, tracheo-bronchial, salivary, and sweat glands, also co-express HDAC8. In addition, we have found that HDAC8 is expressed by myofibroblasts residing in some, but not all, myofibroblasts containing normal human tissues tested in this study. Indeed, we have observed that the enzyme is present in lung alveolar septae myofibroblasts, prostate stromal cells, reticular cells of the spleen, external theca cells of the ovary, testis peritubular myoid cells, and intestine subepithelial myofibroblasts. Interestingly, we have been able to show that these myofibroblastic cells co-express {alpha}-SMA and smooth muscle myosin, as previously reported.48-54 On the other hand, no detectable level of HDAC8 expression has been observed in reticular cells of the thymus, stromal cells of the breast, periacinar stellate cells of the pancreas, perisinusoidal stellate (Ito) cells of the liver, and mesangial cells of the kidney. It is noteworthy that these HDAC8-negative myofibroblasts may express neither {alpha}-SMA nor SMMHC.40,43,46,55-61 Therefore, altogether these observations suggest that co-expression of HDAC8, {alpha}-SMA, and SMMHC may identify subsets of myofibroblasts residing in specific normal human tissues.

Our original observations also include that HDAC8, a class I HDAC, is predominantly detected in the cytoplasm of smooth muscle cells rather than in the nuclear compartment, both in vivo and in vitro. The prominent cytosolic expression of HDAC8 observed in this current study contrasts with a previous observation showing that a N-terminally myc-tagged HDAC8 construct transiently transfected into NIH-3T3 cells was expressed only in the cell nucleus.10 These differences in protein subcellular localization may be explained by an improper folding of the N-terminally tagged protein construct, which may impede its localization to the cytoplasm. Indeed, another group has found the enzyme to be located in the nucleus as well as in the cytoplasm of HEK293 cells transfected with an HDAC8 construct tagged at the C terminus.11 In our study, NIH-3T3 cells transfected with a C-terminally V5-tagged HDAC8 construct expressed the exogenous protein both in the cytoplasm and in the nucleus. This subcellular distribution of exogenous HDAC8 was similar to that of the endogenously expressed enzyme in these cells, as revealed by nuclear and cytoplasmic fractionation and immunocytochemistry experiments. We therefore conclude that HDAC8 is a predominantly cytosolic HDAC. This observation is not unprecedented for a class I HDAC because HDAC3 can be also cytoplasmic and contains a nuclear export signal in its central portion.33,62 Whether HDAC8 also possesses a nuclear export motif remains to be determined.

Contradictory results have been reported with respect to the capacity of HDAC8 to deacetylate histones. Hu and colleagues10 have shown that HDAC8 expressed in HEK293 cells, could exhibit TSA-inhibitable deacetylase activity toward acetylated histones. In addition, they have observed in co-transfection experiments that HDAC8 was able to repress a viral SV40 early promoter activity. Finally, they have shown that bacterially expressed, purified human recombinant HDAC8 protein was active toward acetylated histones H3 and H4, suggesting that HDAC8 may be active in the absence of cofactors and without further posttranslational modifications that occur in eukaryotic cells. Buggy and colleagues13 have shown that purified FLAG-tagged human HDAC8 from transfected insect Sf9 cells could deacetylate a radioacetylated histone H4 peptide in a reaction inhibited by both sodium butyrate and TSA. Van den Wyngaert and colleagues11 created HEK293 clones that constitutively overexpressed HDAC8 fivefold to sixfold above untransfected cells. Total cell extracts from HDAC8 stably transfected cells showed increased deacetylation activity as compared with the vector-transfected control cells. However, no change in the level of histone acetylation was observed between the HDAC8- and empty vector-transfected cells. In addition, immunoprecipitated HDAC8 showed no significant deacetylase activity. Whether HDAC8 has HDAC activity remains to be definitively clarified. However, our findings showing a prominent cytoplasmic localization of the enzyme suggest that HDAC8 may have other substrate(s) than acetylated histones. In addition, cytoplasmic HDAC8 distribution with a filamentous-like pattern reminiscent of stress fibers was suggestive of a cytoskeletal association. Further to this observation, we have used double-immunofluorescence staining experiments coupled with confocal microscopy analysis to show that epitope-tagged HDAC8 overexpressed in NIH-3T3 cells forms cytoplasmic stress fiber-like structures that co-localize with {alpha}-SMA. Recently, Durst and colleagues63 have demonstrated that the protein product resulting from inversion(16), a frequent chromosomal translocation found in acute myeloid leukemia, which fuses the first 165 amino acids of core binding factor ß (CBF-ß) to the tail region of SMMHC, specifically associates with HDAC8 through a domain present in the C-terminal SMMHC portion of the CBF-ß-SMMHC fusion protein. Moreover, it has been previously shown that, in NIH-3T3 cells transfected with full length CBF-ß-SMMHC plasmid cDNA, the fusion protein was present in cytoplasm stress fibers-like structures co-localizing with actin filaments.64 Altogether, these reports as well as our findings suggest that HDAC8 may be involved in the regulation of smooth muscle acto-myosin complexes. Additional studies are required to test this hypothesis. In this respect, it is noteworthy that HDAC6, a class II HDAC, and SIRT2, a class III HDAC, have been recently reported to deacetylate cytoskeletal acetylated {alpha}-tubulin.28,45,65,66

In conclusion, our results demonstrate that HDAC8, a predominantly cytoplasmic HDAC, is a novel marker of smooth muscle cell differentiation. We are conducting further investigations to determine the potential involvement of HDAC8 in the regulation of the smooth muscle contractile apparatus.


   Acknowledgements
 
We thank Pascale Heneaux and Marie-Christine Petit for their expert technical assistance.


   Footnotes
 
Address reprint requests to David Waltregny, M.D., Ph.D., Metastasis Research Laboratory, Pathology Building, Bat. B23, level –1, CHU Sart Tilman Liège, B-4000 Liège 1, Belgium. E-mail: david.waltregny{at}ulg.ac.be

Supported by the National Fund for Scientific Research (Belgium), the Centre Anti-Cancéreux de l’Université de Liège, the Fondation Léon Frédéricq, TELEVIE, and the Interuniversity Attraction Pole.

Accepted for publication April 21, 2004.


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