SARA in TGF-ß1 Signaling
SARA was initially described as a recruitment factor, bringing R-Smads to the TßR complex for phosphorylation (Tsukazaki et al. 1998). In that study, SARA and Smad2 were described as being associated basally but separated upon TGF-ß stimulation. However, a subsequent study found minimal basal association between SARA and Smad2, but a strong interaction after treatment with TGF-ß (Runyan et al. 2005). SARA can associate basally with TßRI and TßRII (Tsukazaki et al. 1998). Therefore, it remains unclear whether SARA is predominantly localized with R-Smads or with TßRs in unstimulated cells.
The SBD of SARA can interact with the MH2 domain of either Smad2 or Smad3 (Tsukazaki et al. 1998) when over-expressed in cultured cell lines. However, examination of endogenous protein expression and interaction suggests that SARA preferentially binds Smad2 over Smad3 (Runyan et al. 2005). The ability of Smad3 to interact with SARA has been reported to be dispensable for Smad3 function (Goto et al. 2001; Runyan et al. 2009). In contrast, knockdown of SARA in HKC kidney proximal tubule cells results in a specific reduction in Smad2 expression, phosphorylation, nuclear translocation, and transcriptional activity (Runyan et al. 2009). In hepatic stellate cells, SARA expression is reduced during myofibroblastic differentiation and this reduced expression of SARA corresponds to a switch in predominant R-Smad activation by TGF-ß from Smad2 to Smad3 (Liu et al. 2003).
In addition to promoting Smad2-dependent signaling, SARA may also function as a negative regulator of the TGF-ß pathway. The Drosophila melanogaster homolog of SARA (Sara) binds the catalytic subunit of the type 1 serine/threonine protein phosphatase (PP1c), and mutations that disrupt this interaction result in hyper-phosphorylated type I receptor and inhibition of TGF-ß-mediated signaling (Bennett and Alphey 2002). Later studies suggested that it was the Smad7-mediated antagonism of TßRI that was enhanced by the SARA PP1c interaction. Smad7 binds to the GADD34 regulatory subunit of the PP1 holoenzyme, which results in recruitment of the PP1c catalytic subunit to dephosphorylate the TßRI. This recruitment is enhanced by SARA interaction with PP1c (Shi et al. 2004).
Endocytosis, SARA, and TGF-ß1 Signaling
Tightly controlled localization of the TGF-ß signaling components makes logistic sense as a modulator of the otherwise simple and linear Smad pathway. However, while roles for clathrin-mediated endocytosis (CME) as well as caveolar internalization have recently been proposed to impact the TGF-ß pathway, receptor trafficking in TGF-ß-mediated Smad signaling is not completely understood.
CME initiates with formation of clathrin-coated vesicles via interaction of cytosolic proteins with components of the inner leaflet of the plasma membrane. Transmembrane receptors bind directly or indirectly to the heterotetrameric AP-2 adaptor complex which then interacts with the clathrin triskelion that polymerizes to form a basket-shaped lattice pulling the membrane into the cell. This invaginated clathrin-coated vesicle (CCV) is then internalized to the major sorting center of the cell, the early endosome. From the early endosome, internalized material can be transported back to the membrane through the recycling endosome, or routed to either the late endosome or to the trans-Golgi network. From either the late endosome (also referred to as a multi-vesicular body) or the trans-Golgi, internalized proteins may be redirected back to the early endosome, back to the membrane, or to the lysosome for degradation. These processes of endocytosis, trafficking, endosome fusion, and exocytosis (reviewed in (Mousavi et al. 2004; Miaczynska et al. 2004)) are controlled by diverse members of the Ras-like small G proteins called Rab GTPases. Like other GTPases, the Rab proteins cycle between an active, GTP-bound, and an inactive, GDP-bound state. Individual isoforms of the Rab proteins are localized to the surface of distinct, membrane-bound organelles. Rab4, Rab5, and Rab11 localize to early endosomes. However, Rab4 or Rab11 may also be found on the surface of perinuclear recycling endosomes. Rab7 and Rab9 localize to late endosomes and lysosomes, and Rab1 and Rab2 associate with vesicles mediating ER-to-Golgi transport (Seachrist and Ferguson 2003).
The FYVE domain in SARA directs its localization to Rab5-containing early endosomes. Within the early endosome, SARA interacts with Smad2 and the TßR complex. Other components that may be part of the SARA-containing endosome are the early endosome antigen 1 (EEA1), the cytoplasmic isoform of the promyelocytic leukemia tumor suppressor (cPML), and the hepatic growth factor-regulated tyrosine kinase substrate (Hrs).
EEA1 is a protein involved in the formation of the early endosome. cPML expression is induced by TGF-ß1 and has been shown to be required for SARA association with Smad2 and for SARA and TßR accumulation in the early endosome (Lin et al. 2004). Hrs is another FYVE domain-containing protein that regulates endosomal sorting and plays a critical role in the recycling and degradation of membrane receptors. Hrs binds to Smad2 and cooperates with SARA to stimulate activin-mediated signaling (Miura et al. 2000).
While the importance of internalization in dampening a signal through downregulation of receptors is well studied, endocytosis may also serve to propagate the signal, and this has led to the concept of a “signaling endosome” (Miaczynska et al. 2004; Benmerah 2004). The importance of endocytosis in TGF-ß1 signaling has been described in numerous reports (Hayes et al. 2002; Penheiter et al. 2002; Runyan et al. 2005). Disruption of SARA endocytic localization through either expression of a mutant SARA lacking the FYVE domain (SARA/Δ1-664) (Tsukazaki et al. 1998) or inhibition of PI3P generation by wortmannin treatment (Itoh et al. 2002) causes a redistribution of SARA from punctate endocytic structures to the cytosol, and can prevent TGF-ß1-mediated transcriptional responses.
Putative Physiological Role of SARA
In developing tissues, morphogens can elicit concentration-dependent responses at very long ranges, and even small variations in their concentration can create very different effects. Because many signaling molecules are associated with intracellular membrane-bound compartments, these compartments, the signaling components, and their activation states need to be equally distributed between daughter cells during cell division. In the developing wing of Drosophila melanogaster, a specialized subset of Sara-endosomes, and the receptors therein, associate with the spindle machinery to segregate into the two daughter cells, thus allowing equal inheritance so that they retain the Dpp signaling levels of the mother cell (Bokel et al. 2006).
SARA has been reported to play a role in the signaling and remodeling events within the bone marrow niche where portions of the hematopoietic progenitor cells (HPC) membrane are actively endocytosed by osteoblasts and delivered to SARA-positive, signaling endosomes. In response to intercellular transfer, osteoblasts exhibit decreased Smad signaling and increased production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HPC homing to bone marrow. Because TGF-ß signaling can downregulate SDF-1, the transferred material within SARA-endosomes may sequester SARA away from its general cofactor function in Smad activation, allowing the osteoblast to produce more SDF-1 (Gillette et al. 2009).
SARA has also been reported to play a role in maintaining epithelial cell phenotype. During embryonic development, cells may initially have multiple characteristics but gradually differentiate into a more quiescent phenotype that has specialized functions. This process, known as mesenchymal-to-epithelial transition (MET), can be reversed in mature tissues in response to injury and attempted tissue repair in the process of epithelial-to-mesenchymal transition (EMT). Should such repair be inappropriate or misregulated, EMT can promote cancer progression and fibrosis. TGF-ß is a well-established mediator of EMT, and at least in renal fibrosis, it may give rise to myofibroblast-like, matrix-producing cells. In a human kidney proximal tubule epithelial cell line, downregulation of SARA expression is a requirement for TGF-ß-induced EMT. This depletion of SARA expression results in reduced Smad2 expression and activity via enhancement of Smad2 interaction with the ubiquitination factor Smurf2 (Runyan et al. 2009). Similar to EMT induced by TGF-ß, SARA expression has been reported to decline during EMT induced by high glucose (Tang et al. 2010). The expression of SARA also declines during myofibroblast-like transition of hepatic stellate cells (Liu et al. 2003) and during progression of liver fibrosis (Tao et al. 2006). Therefore, SARA expression may be protective against fibrosis where EMT is a contributor.
Other disease models suggest an increase in SARA expression. In asthma, damage to the bronchial epithelium results in release of TGF-ß, which can suppress EGF-induced proliferation of epithelial cells, and higher levels of SARA expression are found when comparing epithelial cells from subjects with asthma to healthy controls (Semlali et al. 2008). Constitutive upregulation of SARA along with other components of the TGF-ß signaling pathway has been found in rheumatoid arthritis compared to osteoarthritis synovial fibroblasts (Pohlers et al. 2007).
SARA is an adaptor protein, which interacts with TGF-ß receptors and R-Smads and localizes to EEA1-containing, Rab5-positive early endosomes through clatherin-mediated endocytosis. While the precise role of SARA in TGF-ß signaling is not fully characterized, it seems that SARA may preferentially modulate the Smad2-dependent response to TGF-ß with little effect on the Smad3-mediated response. The importance of SARA’s ability to localize to early endosomes has been demonstrated by a number of studies. However, the exact role of endocytosis in TGF-ß signaling is not known. Although there are few studies linking SARA to a physiologic or disease process, it appears that SARA plays a negative role in fibrosis associated with its ability to suppress EMT and help maintain the intact epithelium.
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