TGF beta-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-beta (TGFbeta) enhanced cartilage repair and whether TGFbeta-induced fibrosis was blocked by local expression of the intracellular TGFbeta inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFbeta. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFbeta-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFbeta and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFbeta restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFbeta-induced an elevation in PG content in cartilage of the OA model. TGFbeta-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFbeta on cartilage. Adenoviral overexpression of TGFbeta stimulated repair of IL-1- and OA-damaged cartilage. TGFbeta-induced synovial fibrosis was blocked by locally inhibiting TGFbeta signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.     

GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor

The cascade of phosphorylation is a pivotal event in transforming growth factor beta (TGFbeta) signaling. Reversible phosphorylation regulates fundamental aspects of cell activity. TGFbeta-induced Smad7 binds to type I receptor (TGFbeta type I receptor; TbetaRI) functioning as a receptor kinase antagonist. We found Smad7 interacts with growth arrest and DNA damage protein, GADD34, a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which subsequently recruits catalytic subunit of PP1 (PP1c) to dephosphorylate TbetaRI. Blocking Smad7 expression by RNA interference inhibits association of GADD34-PP1c complex with TbetaRI, indicating Smad7 acts as an adaptor protein in the formation of the PP1 holoenzyme that targets TbetaRI for dephosphorylation. SARA (Smad anchor for receptor activation) enhances the recruitment PP1c to the Smad7-GADD34 complex by controlling the specific subcellular localization of PP1c. Importantly, GADD34-PP1c recruited by Smad7 inhibits TGFbeta-induced cell cycle arrest and mediates TGFbeta resistance in responding to UV light irradiation. The dephosphorylation of TbetaRI mediated by Smad7 is an effective mechanism for governing negative feedback in TGFbeta signaling.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.     

The balance between acetylation and deacetylation controls Smad7 stability

Transforming growth factor beta (TGFbeta) regulates multiple cellular processes via activation of Smad signaling pathways. We have recently demonstrated that the inhibitory Smad7 interacts with the acetyl transferase p300 and that p300 acetylates Smad7 on two lysine residues. These lysine residues are critical for Smurf-mediated ubiquitination of Smad7, and acetylation protects Smad7 from TGFbeta-induced degradation. In this study we demonstrate that Smad7 interacts with specific histone deacetylases (HDACs) and that the same HDACs are able to deacetylate Smad7. The interaction with HDACs is dependent on the C-terminal MH2 domain of Smad7. In addition, HDAC1-mediated deacetylation of Smad7 decreases the stability of Smad7 by enhancing its ubiquitination. Thus, our results demonstrate that the degradation of Smad7 is regulated by the balance between acetylation, deacetylation and ubiquitination, indicating that this could be a general mechanism to regulate the stability of cellular proteins.     

Role of SIRT1 in homologous recombination

The class III histone deacetylase (HDAC) SIRT1 plays a role in the metabolism, aging, and carcinogenesis of organisms and regulates senescence and apoptosis in cells. Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. However, its exact functions in DNA repair remained elusive. Using nuclear foci analysis and fluorescence-based, chromosomal DSB repair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis. Interestingly, we demonstrate that inactivation of Rad51 does not eliminate the effect of SIRT1 on HR. By epistasis-like analysis through knockdown and use of mutant cells of distinct SIRT1 target proteins, we show that the non-homologous end joining (NHEJ) factor Ku70 as well as the Nijmegen Breakage Syndrome protein (nibrin) are not needed for this SIRT1-mediated effect, even though a partial contribution of nibrin cannot be excluded. Strikingly however, the Werner helicase (WRN), which in its mutated form causes premature aging and cancer and which was linked to the Rad51-independent single-strand annealing (SSA) DSB repair pathway, is required for SIRT1-mediated HR. These results provide first evidence that links SIRT1's functions to HR with possible implications for genomic stability during aging and tumorigenesis.     

Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.     

The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction

Signaling from the activin/transforming growth factor beta (TGFbeta) family of cytokines is a tightly regulated process. Disregulation of TGFbeta signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFbeta-signaling cascade. TGFbeta-induced expression of GRK2 acts in a negative feedback loop to control TGFbeta biological responses. Upon TGFbeta stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFbeta-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFbeta-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction.     

SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells by controlling p53 subcellular localization

Nuclear tumor suppressor p53 transactivates proapoptotic genes or antioxidant genes depending on stress severity, while cytoplasmic p53 induces mitochondrial-dependent apoptosis without gene transactivation. Although SIRT1, a p53 deacetylase, inhibits p53-mediated transactivation, how SIRT1 regulates these p53 multifunctions is unclear. Here we show that SIRT1 blocks nuclear translocation of cytoplasmic p53 in response to endogenous reactive oxygen species (ROS) and triggers mitochondrial-dependent apoptosis in mouse embryonic stem (mES) cells. ROS generated by antioxidant-free culture caused p53 translocation into mitochondria in wild-type mES cells but induced p53 translocation into the nucleus in SIRT1(-/-) mES cells. Endogenous ROS triggered apoptosis of wild-type mES through mitochondrial translocation of p53 and BAX but inhibited Nanog expression of SIRT1(-/-) mES, indicating that SIRT1 makes mES cells sensitive to ROS and inhibits p53-mediated suppression of Nanog expression. Our results suggest that endogenous ROS control is important for mES cell maintenance in culture.