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Histone deacetylase HDAC8 associates with smooth muscle -actin and is essential for smooth muscle cell contractility

David Waltregny^*,,1,2, Wendy Glénisson^*,1, Siv Ly Tran^*, Brian J. North, Eric Verdin, Alain Colige and Vincent Castronovo^*

^* Metastasis Research Laboratory,
Department of Urology,
Laboratory of Connective Tissues Biology, University of Liège, Liège, Belgium,
Gladstone Institutes for Virology and Immunology, University of California, San Francisco, California, USA

² Correspondence: University of Liège, Pathology Building, level-1, Bat. B23, Liege B-4000, Belgium. E-mail: david.waltregny{at}ulg.ac.be

SPECIFIC AIMS

HDAC8, a class I histone deacetylase, is expressed exclusively in vivo by cells showing smooth muscle differentiation. This enzyme exhibits a prominent cytoplasmic localization in tissues and in cultured human smooth muscle cells (HSMCs), where it displays a cytoskeleton-like pattern of distribution reminiscent of actin stress fibers and is coexpressed with smooth muscle -actin (-SMA) and smooth muscle myosin heavy chain (SMMHC). We tested whether HDAC8 associates with the smooth muscle cytoskeleton and sought to examine the possible functions of HDAC8 in smooth muscle cell biology.

PRINCIPAL FINDINGS

1. HDAC8 associates with -SMA in vitro and in vivo
Two approaches were used to investigate the existence of an association between HDAC8 and the actin cytoskeleton in HSMCs. We first performed cell fractionation experiments in which sequential detergent and salt extractions produced three fractions: one cytosolic in origin ("free cytosolic"), one enriched in cytoskeletal components ("cytoskeletal bound"), and one containing intracellular proteins associated with subcellular organelle remnants ("membrane bound"). After copurification, the remaining insoluble pellet was subjected to F-actin depolymerization and the supernatant was collected as the "solubilized pellet." As assessed by immunoblot, -SMA and ß-actin isoforms were present in all three fractions, with an expected modest enrichment in the cytoskeletal-bound fraction. HDAC8 was found in the same fractions, with slightly higher levels observed in the cytoskeletal-bound fraction. HDAC8 and (as expected) the actin isoforms copurified in the solubilized pellet fraction. In contrast, two other class I HDACs, HDAC1 and HDAC3, were absent from the cytoskeletal-bound and solubilized pellet fractions and were detectable only in the membrane-bound extracts. Therefore, under these experimental conditions, HDAC8 but not HDAC1 or HDAC3 appeared to be associated with components of the cytoskeleton.

We then tested whether HDAC8 could associate, directly or indirectly, with -SMA. Endogenous -SMA was immunoprecipitated from HSMC total protein lysates and immunoprecipitates were subjected to immunoblot using antibodies directed against -SMA, HDAC8, HDAC1, and HDAC3. HDAC8 coimmunoprecipitated with -SMA whereas HDAC1 and HDAC3 were not detected in the immunocomplexes. Similar pull-down assays carried out with the use of a monoclonal anti-ß-actin antibody failed to detect -SMA, HDAC8, or HDAC3 in the ß-actin-containing immunocomplexes.

Coimmunoprecipitation experiments were performed using protein lysates from normal human prostate tissues, known to be highly enriched in HDAC8-expressing myofibroblasts. As in HSMCs, endogenous HDAC8, but not HDAC1 or HDAC3, associated with -SMA in normal prostate tissue (Fig. 1 ). The -SMA-containing immunocomplexes were devoid of detectable ß-actin (Fig. 1) . Coimmunoprecipitation using anti-ß-actin antibodies and prostate lysates did not yield detectable HDAC8 or -SMA in the immunoprecipitates.

View larger version (41K): [in this window] [in a new window]   Figure 1. Interaction of HDAC8 with -SMA in normal human prostate tissues. Normal human prostate tissues were lysed in ice-cold low stringency buffer (LB) or in high stringency buffer (HB). Protein lysates were immunoprecipitated with a monoclonal antibody against -SMA or IgG2a. Whole extracts and immunocomplexes were subjected to immunoblot analysis using antibodies directed against -SMA, ß-actin, HDAC8, HDAC1, and HDAC3.

2. Contraction of type I collagen lattices by HSMCs requires HDAC8
To investigate whether HDAC8 may be involved in regulating smooth muscle contractile apparatus, we have used RNA interference to determine the effect of a reduction of HDAC8 abundance in HSMCs on the ability of these cells to contract type I collagen lattices in vitro. HSMCs were transfected with two siRNAs specific for HDAC8: HDAC8 siRNA#1 and HDAC8 siRNA#2. HDAC8 siRNA#1 transfection induced substantial silencing of HDAC8 expression. However, transfections with HDAC8 siRNA#2 were inefficient in reducing HDAC8 abundance in these cells. We thus used HDAC8 siRNA#2 as an additional negative control together with mock transfections carried out without siRNAs in the transfection medium. We showed that HDAC1 and HDAC3 expression levels as well as those of ß-actin and -tubulin were not reduced by HDAC8 siRNA#1 transfections in NIH-3T3 cells or HSMCs, suggesting this siRNA induced the specific degradation of HDAC8 transcripts.

To test whether HDAC8 may participate in conferring contractile capacity to smooth muscle cells, HSMCs were transfected without siRNA or with HDAC8 siRNA#1, HDAC8 siRNA#2, or HDAC6 siRNA. Forty-eight hours after two transfections, HSMCs were lysed in protein extraction buffer for immunoblot analysis and assayed for collagen gel contraction or reseeded on plastic dishes to evaluate their morphology. The amount of HDAC8 was decreased only in cells transfected with HDAC8 siRNA#1. Only HSMCs transfected with HDAC6 siRNAs displayed increased levels of acetylated -tubulin whereas the abundance of HDAC8 or acetylated -tubulin was unchanged in mock or HDAC8 siRNA#2-transfected HSMCs. Figure 2 A shows representative images of collagen lattices photographed 24 h after relaxation. At that point, mean area of the lattices containing HDAC8 siRNA#1-transfected HSMCs was significantly larger than that of lattices incubated with mock-, HDAC8 siRNA#2-, or HDAC6-transfected HMSCs (Fig. 2B ).

View larger version (48K): [in this window] [in a new window]   Figure 2. HDAC8 silencing by RNA interference impairs the capacity of HSMCs to contract type I collagen lattices. HSMCs transfected with or without siRNAs for HDAC8 or HDC6 were added to a collagen solution. Samples of the cell/collagen mixtures were placed in plastic dishes and incubated at 37°C. Relaxation of the polymerized gels was initiated 1 h after incubation. Gel contraction was allowed to proceed for 144 h. A minimum of 3 lattices was assayed per experimental condition. A) Representative image of collagen lattices photographed 24 h after relaxation of the polymerized lattices. White dashed circular lines show the circumference of the collagen gels. B) The area of the gels was measured at defined time intervals. The area of contracted lattices was calculated as a % of the surface of the dish. Mean values are expressed ± SD of the mean. Experiments were performed at least 3 times, with similar results.

3. HDAC8 silencing by RNA interference induces morphological changes in HSMCs
Morphology of siRNA-transfected HSMCs was assessed before and after reseeding, when transfected HSMCs were added into the collagen matrices. Before trypsinization and reseeding, no change in cellular morphology was noted by light microscopy examination. HDAC8 siRNA#2- and HDAC6 siRNA-transfected HSMCs exhibited no obvious modification in cell shape or size compared with mock-transfected HSMCs at any time after reseeding. Strikingly, HDAC8 siRNA#1-transfected HSMCs exhibited a noticeable reduction in size, with decreased cell spreading. These morphological changes were noted as early as 1 h after replating and were maintained for 1 wk. Thereafter, cellular morphology resumed to that of control HSMCs.

CONCLUSIONS AND SIGNIFICANCE

Our recent demonstration that HDAC8 is detected in the cytosol of normal human cells showing smooth muscle differentiation has suggested potential new functions for this enzyme. This study provides the first evidence that a class I HDAC associates with the actin cytoskeleton. Initial cellular fractionation experiments have shown that, in contrast to HDAC1 and HDAC3, HDAC8 can be copurified with -SMA and ß-actin and can be released from detergent-insoluble pellets by F-actin depolymerization. These results suggest that this HDAC may associate at least in part with the actin cytoskeleton, possibly with its filamentous form (F-actin), as already suggested by our earlier colocalization studies. Our results from coimmunoprecipitation experiments indicate that HDAC8 associates with the smooth muscle isoform of -actin in primary human smooth muscle cells, NIH-3T3 cells, and human prostate tissue. No association has been found between HDAC8 and the ubiquitously expressed ß-actin isoform. Neither HDAC1 nor HDAC3, two other class I HDACs, were detected in the -SMA-containing immunocomplexes, further suggesting that HDAC8 uniquely and specifically interacts with the smooth muscle cytoskeleton.

Whether HDAC8 interacts directly or indirectly with -SMA and whether any smooth muscle cytoskeletal protein may be a target of HDAC8’s deacetylase activity remain to be elucidated. Several lines of evidence currently suggest that histones may not be the "natural" substrates of HDAC8. It has been reported that total cell extracts from HDAC8 stably transfected HEK293 clones overexpressing HDAC8 5- to 6-fold above untransfected cells do not exhibit any change in the level of histone acetylation vs. that of empty vector-transfected cells. HDAC8 immunoprecipitated from these cells exhibits no significant deacetylase activity. A newly identified potent HDAC8 selective inhibitor, SB-379872-A, is unable to increase cellular histone acetylation or the activity of an early SV40 promoter.

HDAC8 RNA interference was used in this study to investigate the potential functions of this enzyme in smooth muscle cell biology. A striking reduction in cell size with decreased spreading was found in HDAC8-silenced HSMCs whereas control and HDAC6 siRNA-transfected HSMCs exhibited no obvious modification in cell shape or size at any time before or after reseeding. HDAC8 silencing-induced alterations of smooth muscle cell shape were readily detectable only after trypsinization and replating of the cells. This suggests that HDAC8, possibly through its interaction with -SMA cytoskeletal protein, regulates the dynamics of smooth muscle cytoskeleton rather than its statics and may be involved in mechanisms responsible for the attachment/spreading of these cells. This involvement may account at least in part for the reduced capability of HDAC8-silenced HSMCs to contract type I collagen lattices. Indeed, we further demonstrated that HDAC8 is necessary for conferring the capacity to HSMCs to contract type I collagen lattices. We speculate that HDAC8 may exert its effects through a predominant cytosolic deacetylase activity possibly affecting the function of smooth muscle cytoskeletal proteins, although we cannot dismiss the possibility that HDAC8 may have gene expression-related activities (Fig. 3 ). Further studies are needed to address this issue. In conclusion, we have demonstrated that HDAC8 associates with and regulates smooth muscle cell cytoskeleton dynamics. Our findings lay the ground for the rational investigation of the mechanisms underlying HDAC8-enabled smooth muscle cytoskeleton remodeling.

View larger version (110K): [in this window] [in a new window]   Figure 3. HDAC8 associates with smooth muscle -actin and is essential for actin smooth muscle cell contractility. HDAC8 interacts physically and colocalizes with -SMA mainly as stress fiber-like structures in the cytosol of human cells showing smooth muscle differentiation (A). Forced repression of HDAC8 expression by RNA interference alters the morphology of human primary smooth muscle cells and their capacity to contract type I collagen lattices, possibly through a predominant cytosolic deacetylase activity of HDAC8 affecting the function of smooth muscle cytoskeletal proteins (B).

FOOTNOTES

¹ These authors contributed equally to this study.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2303fje; doi: 10.1096/fj.04-2303fje

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