Reversible acetylation of alpha-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated alpha-tubulin is tightly controlled and stereotypic. Acetylated alpha-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it associates with microtubules and localizes with the microtubule motor complex containing p150(glued) (ref. 3). In vivo, the overexpression of HDAC6 leads to a global deacetylation of alpha-tubulin, whereas a decrease in HDAC6 increases alpha-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates alpha-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biological processes beyond histone metabolism and gene transcription.

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in the vast majority of cellular processes and is fundamentally important during the entire life of all metazoans. Deregulation of TGF-β/BMP activity almost invariably leads to developmental defects and/or diseases, including cancer. The proper functioning of the TGF-β/BMP pathway depends on its constitutive and extensive communication with other signaling pathways, leading to synergistic or antagonistic effects and eventually desirable biological outcomes. The nature of such signaling cross-talk is overwhelmingly complex and highly context-dependent. Here we review the different modes of cross-talk between TGF-β/BMP and the signaling pathways of Mitogen-activated protein kinase, phosphatidylinositol-3 kinase/Akt, Wnt, Hedgehog, Notch, and the interleukin/interferon-gamma/tumor necrosis factor-alpha cytokines, with an emphasis on the underlying molecular mechanisms.

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The transforming growth factor ␤s (TGF-␤s) are a group of multifunctional growth factors that inhibit cell cycle progression in many cell types. The TGF-␤-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-␤ on both the levels and activities of the G 1 cyclins and their cyclin-dependent kinase partners. The ability of TGF-␤ to inhibit the activity of these kinase complexes derives in part from its regulatory effects on the cyclin-dependent kinase inhibitors, p21/WAF1/Cip1, p27 Kip1, and p15. Upon treatment of cells with TGF-␤, these three inhibitors bind to and block the activities of specific cyclin-cyclin-dependent kinase complexes to cause cell cycle arrest. Little is known, however, on the mechanism through which TGF-␤ activates these cyclin-dependent kinase inhibitors. In the case of p21, TGF-␤ treatment leads to an increase in p21 mRNA. This increase in p21 mRNA is partly due to transcriptional activation of the p21 promoter by TGF-␤. To further define the signaling pathways through which TGF-␤ induces p21, we have performed a detailed functional analysis on the p21 promoter. Through both deletion and mutation analysis of the p21 promoter, we have defined a 10-base pair sequence that is required for the activation of the p21 promoter by TGF-␤. In addition, this sequence is sufficient to drive TGF-␤-mediated transcription from a previously nonresponsive promoter. Preliminary gel shift assays demonstrate that this TGF-␤ responsive element binds specifically to several proteins in vitro. Two of these proteins are the transcription factors Sp-1 and Sp-3. These studies represent the initial steps toward defining the signaling pathways involved in TGF-␤-mediated transcriptional activation of p21.The transforming growth factor ␤s (TGF-␤s), 1 a group of protein hormones that regulate many cellular functions, inhibit cell proliferation by causing growth arrest in the G 1 phase of the cell cycle (1-4). Progression through G 1 is dependent on the sequential formation, activation, and subsequent inactivation of the G 1 cyclin-cyclin-dependent kinase complexes, primarily cyclin D-cyclin-dependent kinase 4 and cyclin E-cyclin-dependent kinase 2 complexes (5, 6). The TGF-␤-induced G 1 cell cycle arrest has been attributed to the regulatory effects of TGF-␤ on both the levels and activities of these G 1 cyclins and cyclin-dependent kinases (7-9). The inhibition of G 1 cyclin-cyclin-dependent kinase complex activity by TGF-␤ is mediated in part through several members of a recently described family of low molecular weight cyclin dependent kinase inhibitors. These cyclin-dependent kinase inhibitors, which include p21/WAF1/ Cip1, p27 Kip1 , p57 Kip2 , p18, p16, and p15, physically associate with their target cyclins, cyclin-dependent kinases, or cyclincyclin-dependent kinase complexes to inhibit their activities (reviewed in Refs. 10 -13). TGF-␤ regulates the activities of three of these cyclin-dependent kinase inhibitor family members: p27 Kip1 , p15, and p21 (reviewed in Refs. 10...

The Smads are a family of nine related proteins which function as signaling intermediates for the transforming growth factor beta (TGF-beta) superfamily of ligands. To discern the in vivo functions of one of these Smads, Smad3, we generated mice harboring a targeted disruption of this gene. Smad3 null mice, although smaller than wild-type littermates, are viable, survive to adulthood, and exhibit an early phenotype of forelimb malformation. To study the cellular functions of Smad3, we generated Smad3 null mouse embryonic fibroblasts (MEFs) and dermal fibroblasts. We demonstrate that null MEFs have lost the ability to form Smad-containing DNA binding complexes and are unable to induce transcription from the TGF-beta-responsive promoter construct, p3TP-lux. Using the primary dermal fibroblasts, we also demonstrate that Smad3 is integral for induction of endogenous plasminogen activator inhibitor 1. We subsequently demonstrate that Smad3 null MEFs are partially resistant to TGF-beta's antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-beta-mediated growth inhibition. We next examined cells in which Smad3 is most highly expressed, specifically cells of immune origin. Although no specific developmental defect was detected in the immune system of the Smad3 null mice, a functional defect was observed in the ability of TGF-beta to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-beta-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-beta and implicate Smad3 as a potential effector for TGF-beta in modulating immune system function.

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