Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling
© Ibarrola et al; licensee BioMed Central Ltd. 2004
Received: 10 November 2003
Accepted: 19 January 2004
Published: 19 January 2004
Transforming growth factor-βs (TGF-βs), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors.
There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (a ssociated m olecule with the SH 3 domain of STAM), an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in J un kinase a ctivation domain b inding protein and proteasomal subunits) domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-β-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-β signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-β.
This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-β signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-β signaling pathway.
TGF-β ligands belong to a large and expanding family of multifunctional cytokines which include the activins, inhibins, bone morphogenetic proteins (BMPs), growth/differentiation factors, Mullerian inhibiting substance and Nodal. Members of this family regulate a broad range of physiological processes, including organogenesis, proliferation, differentiation, adhesion, motility and apoptosis [1, 2]. Mutations in downstream components of the TGF-β signaling pathways have been implicated in several human diseases .
TGF-β superfamily members signal through a receptor complex formed by the association of two transmembrane proteins designated I and II, each possessing serine/threonine kinase activity (for reviews see [1, 4, 5]). TGF-β binding induces the assembly of the type I and type II receptors [6, 7], thereby enabling the type II receptor to phosphorylate and activate the type I receptor [8, 9]. Activated type I receptors then phosphorylate Smad2 and Smad3 receptor-regulated Smad proteins (R-Smads) at the carboxy terminal motif SSxS [10, 11]. Phosphorylated R-Smads interact with Smad 4, the unique Co-Smad [11–13]. This complex accumulates in the nucleus where it regulates transcriptional activity of several genes in association with transcriptional activators and corepressors (for reviews see [14–17]).
The specific transcriptional outcome in response to TGF-β is achieved by the interplay of intracellular regulators at different levels. Within the Smad family of proteins, Smad6 and 7 (I-Smads) negatively regulate TGF-β signaling [18–20]. Smad6 also inhibits BMP signaling, while activin signaling is negatively regulated by Smad7 . In addition to the Smad proteins, other regulatory molecules work as adapter and/or scaffolding proteins, such as SARA, that mediates the access of Smads to activated receptors . Finally, the specific outcome of TGF-β signaling is modulated by several other signaling pathways (for review see [15, 23]).
The human unidentified gene-encoded large proteins database (HUGE) contains long cDNAs and is a valuable tool to discover new proteins, and to predict structural features with a possible functional relevance [24, 25]. Through the analysis of the cDNA clones contained in this database, we have identified a novel protein that is highly similar to AMSH (a ssociated m olecule with the SH 3 domain of STAM) . Systematic analysis using specific reporters for several pathways showed that this novel molecule, designated AMSH-2, positively regulates TGF-β signaling system but not the TNF signaling pathway.
Results and discussion
Identification of a novel signaling molecule, AMSH-2
Expression of AMSH-2
We corroborated the mRNA distribution of AMSH-2 by performing a BLAST search against the EST database. We found ESTs corresponding to AMSH-2 mRNA in brain, testes, bone, pancreas, fetal liver, kidney, colon, stomach, bone marrow, placenta, breast, pectoral muscle, hypothalamus, ovary as well as in a number of cell lines of different origins, including T cell leukemia, NT2 neuronal and B-cell CLL . These data indicate that, like AMSH , AMSH-2 mRNA is widely expressed.
We raised an antibody against the carboxy terminus of AMSH-2 and measured the expression of AMSH-2 protein in different cell lines. For this purpose, HepG2 and 293T cells lines were metabolically labeled with 35S and cell lysates were immunoprecipitated with preimmune or anti-AMSH-2 antiserum. We observed a band with a molecular weight of approximately 56 kDa, which is in agreement with the predicted molecular weight of AMSH-2. This band was specific for AMSH-2 as it was absent in the samples immunoprecipitated with pre-immune serum (Figure 2B).
AMSH-2 enhances gene expression of TGF-β-dependent promoters
In order to study the functional role of AMSH-2, we constructed several epitope-tagged versions of AMSH-2. The entire open reading frame of AMSH-2 was subcloned by PCR into pEF mammalian expression vectors that provide a Myc or V5 epitopes at the C-terminus. Ectopic expression of AMSH-2/Myc was undertaken and confirmed by metabolic labeling of 293T cells, followed by immunoprecipitation with anti-Myc antibodies (Figure 2C).
To account for possible non-specific effects, we tested the effect of AMSH-2 overexpression on the TNF signaling pathway. We used an ELAM-luciferase reporter construct as a reporter for activation of this pathway. This reporter contains the ELAM-1 promoter (-730 to +52) and is well known to be induced by TNF stimulation . We therefore cotransfected 293 cells with ELAM-luciferase and either AMSH-2, empty vector or TRAF2, a prototypical member of the TRAF family of adapter proteins known to potentiate TNF signaling pathway [38–40]. We did not observe any increase in the luciferase levels in AMSH-2 transfected cells as compared with the empty vector, although overexpression of TRAF2 led to a significant upregulation of the promoter (Figure 3C). Taken together, these results imply that the AMSH-2 regulates TGF-β signaling pathway.
AMSH-2 negates the inhibitory effect of Smad7 on TGF-β signaling
Because AMSH-2 interacts with Smad7, we also analyzed the effect of AMSH-2 on the ability of Smad7 to inhibit TGF-β signaling in HepG2 cells. As expected, overexpression of Smad7 attenuated the induction of p3TP-luciferase activity by TGF-β (Figure 4B). Interestingly, overexpression of AMSH-2 negated the inhibitory effect of Smad7 on p3TP expression stimulated by TGF-β (Figure 4B). Therefore AMSH-2 can rescue the inhibitory effects of Smad7 on TGF-β signaling pathway. Given the sequence similarity between AMSH and AMSH-2, it is indeed possible that AMSH-2 also interacts with Smad6. Smad6 has been shown to inhibit the TGF-β pathway  and AMSH is able to rescue the inhibitory effect of Smad6 on BMP signaling pathway . If AMSH-2 is able to interact with Smad6, the effect of AMSH-2 on TGF-β signaling pathway could be explained by its interaction with both Smad7 and Smad6.
AMSH-2 is a positive regulator of TGF-β-dependent transcriptional activation
Our results indicate that AMSH-2 is a positive regulator of TGF-β signaling. This effect might be mediated through the interaction with the inhibitory Smad7 as AMSH-2 is capable of negating the inhibitory effects of Smad7 in TGF-β signaling pathway. Further, use of siRNA to knock down the expression of AMSH-2 reduces signaling by TGF-β. Similar to our studies, it has been suggested that AMSH might be involved in TGF-β signaling. AMSH can interact with both I-Smads (i. e. Smads 6 and 7) and is a positive regulator of BMP signaling pathway . The high degree of homology between these paralogs suggests that AMSH-2 might also interact with Smad6, and therefore might regulate BMP signaling. Although further research needs to be conducted to elucidate the exact mechanism of action of AMSH-2, it is clear that AMSH and AMSH-2 belong to a new family of molecules that positively regulate TGF-β signaling pathway.
cDNAs and constructs
A human cDNA clone [Genbank AB037794] was used as described [24, 25]. To generate wild type AMSH-2 expression vector with a carboxy-terminal Myc epitope, we subcloned AMSH-2 open reading frame into the Nco I and Xho I sites of pEF/myc/cyto (Invitrogen, Carsbad, CA) by standard PCR procedures using the primers: AAAACCATGGATCAGCCTTTTACTGTGAATTC (5' primer) and AAACTCGAGCTGTTCAGATGGTGATGATGAC (3' primer). To generate AMSH-2 V5 carboxy-terminal-tagged expression vector, the open reading frame of AMSH-2 was subcloned into Nco I and Xho I sites of pEntr4 (Invitrogen) using the same 5' primer as described above and AAACTCGAGAGCTGTTCAGATGGTGATGATGACCTAG (3' primer), and transferred to pEF1/V5-HisB (Invitrogen), which has been previously made gateway compatible by inserting a cassette in the EcoRV site.
3TP-lux, PAI-1-luc, pELAMP-luc+, TRAF2, Flag-tagged Smad7, Smad3, have been previously described [6, 20, 36–38, 45] while Flag-tagged Smad2 and HA-tagged Smad4 were generously provided by Joan Massague.
Northern blot analysis
Human tissue northern blot II and cancer cell line blot were obtained from CLONTECH (Palo Alto, CA). We isolated and radiolabeled an AMSH-2 cDNA (nucleotides 1088–2010) produced by digestion of AMSH-2 cDNA clone with HindIII and SphI. Hybridization signals were detected on a BAS-2000 bioimaging analyzer (Fuji film, Tokyo, Japan). After exposure, the blots were stripped and reprobed with beta-actin to control for equal amounts of PolyA+ RNA loading.
Cell culture and antibodies
293 and 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and penicillin/streptomycin. HepG2 cells were grown in MEM supplemented with 10% fetal bovine serum, non essential amino-acids, sodium pyruvate and penicillin/streptomycin.
The peptide corresponding to the C-terminus of human AMSH-2 was synthesized by Boston Biomolecules, Inc (Woburn, MA). The specific AMSH-2 rabbit polyclonal antibody (anti-AMSH-2) was raised at Covance Research Products Inc. (Denver, PA). The following antibodies were obtained: (i) c-Myc mAb 9E10 (Covance Research Products Inc);(ii) anti-V5 and anti-V5 HRP antibodies (Invitrogen); (iii) anti-Flag HRP (UPSTATE, Lake Placid, NY);and (v) anti-HA 12CA5, anti-HA HRP (Roche Diagnostics Corp, Indianapolis, IN).
Metabolic labeling and immunoprecipitation
To test endogenous expression of AMSH-2, 293T and HepG2 cells were metabolically labeled with 35S methionine and 35S cysteine and lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) with 1 mM sodium orthovanadate and protease inhibitors. Samples were incubated either with pre-immune serum or AMSH-2 antiserum. Immunoprecipitated proteins were resolved by SDS-PAGE. To test expression of the Myc-tagged AMSH-2 construct, cells were transfected with 15 μg of DNA using the standard calcium phosphate method. Twenty-four h postransfection, the cells were metabolically labeled for 16 h. and subsequently lysed and immunoprecipitated with anti-Myc antibody as above.
HepG2 cells were transfected with 0.5 μg of the corresponding luciferase reporter, 50 ng of β-galactosidase reporter and 4.5 μg of DNA. Twenty-four h postransfection, the cells were depleted from serum and stimulated for 16–20 h with 5 ng/ml of purified recombinant human TGF-β1 (R&Dsystems. Inc. Minneapolis, MN). Luciferase and β-galatosidase activities were measured according to manufacturer's instructions (Tropix, Bedford, MA). Measurements were corrected by β-galactosidase activity. The experiments were repeated at least three times.
293T cells were cotransfected with 13 μg of AMSH-2 V5 construct and 2 μg of the corresponding Smad constructs. After 48 h, the cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1% NP-40) with 1 mM sodium orthovanadate and protease inhibitors. Lysates were incubated with anti-V5 antibody, and the resulting immunocomplexes were separated in SDS-PAGE. Blots were probed with the indicated HRP conjugated antibody.
To target AMSH-2 and Smad 7, we designed 21 nt siRNA duplexes: AACAATTCCTTGCTGAATGTA and AACCGCAGCAGTTACCCCATC, according to . All siRNA duplexes including lamin A/C siRNA duplexes and scrambled siRNA were obtained from Dharmacon Research. Inc. (Lafayette, Colorado).
HepG2 cells grown in 6 well plates were transfected with 250 ng of 3TP-lux reporter, 50 ng of β-galactosidase reporter and 0.12 nmol of the corresponding siRNA duplex. After 48 h of transfection cells were depleted from serum and treated with 5 ng/ml TGF-β1. Luciferase and β-galactosidase activities were measured after 17 h stimulation.
We thank Harvey Lodish for support and encouragement. Akhilesh Pandey is supported by a Career Development Award from the Breast Cancer SPORE (CA 88843) at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins and by a Sidney Kimmel Scholar Award. Work at the Center of Experimental BioInformatics (CEBI) is supported by a generous grant from the Danish National Research Foundation. N.I was supported by a grant from Ministerio de Ciencia y Technologia (Spain).
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