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Proc Natl Acad Sci U S A. Oct 7, 2008; 105(40): 15417–15422.
Published online Sep 29, 2008. doi:  10.1073/pnas.0807763105
PMCID: PMC2563117
Cell Biology

The microRNA miR-8 is a conserved negative regulator of Wnt signaling

Abstract

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

Keywords: adipogenesis, TCF, Wntless

Wnts are a family of highly conserved, secreted glycoproteins that act at a short range to regulate cell fate decisions during animal development (1, 2). The protein Armadillo (Arm; β-catenin in vertebrates) is central to the highly conserved signaling pathway by which cells respond to the Drosophila protein Wingless (Wg) and other Wnts. In the absence of the Wg/Wnt signal, a cytosolic pool of Arm/β-catenin is phosphorylated and targeted for degradation. Wg/Wnt signaling inhibits this degradation, resulting in stabilization and nuclear translocation of Arm/β-catenin. In the nucleus, Arm/β-catenin forms a complex with members of the TCF/LEF family of transcription factors to regulate gene transcription. Wg/Wnt signaling is highly regulated, and inappropriate activation or inhibition of Wg/Wnt signaling results in developmental defects and diseases in humans, including colorectal cancer and inherited bone diseases (1, 2). Studies that add to our understanding of how the Wg/Wnt pathway is regulated are important, considering the key roles that this pathway plays in animal development and disease.

MicroRNAs are recently discovered regulators that influence cell physiology. These small (21–22 nt), noncoding RNAs posttranscriptionally silence gene expression in animals and plants by binding to specific mRNAs (3, 4). In animals, microRNAs generally bind to the 3′UTR of their mRNA targets and silence gene expression by causing degradation, decreased stability, or translational inhibition of target mRNAs. Hundreds of microRNAs have been identified, most of which are predicted to target multiple mRNAs, suggesting that microRNAs may function as part of an extensive gene regulatory network (3). Indeed, the regulation of conserved developmental signaling pathways (e.g., the Notch, Hedgehog, and TGFβ pathways) by microRNAs has been reported (58). Activation of the Wg/Wnt pathway by the microRNA miR-315 was also been reported recently (9). Expression of miR-315 activates the pathway by targeting the negative regulators Axin and Notum.

Here, we report the identification of a microRNA, miR-8, in a genetic screen for antagonists of Wg signaling. We found that miR-8 inhibits Wg signaling in vivo and in cell culture by targeting the Wg pathway at multiple levels. We demonstrate that miR-8 inhibits TCF protein expression and directly targets two positive regulators of the pathway, wntless (wls) and a gene, CG32767. These data illustrate how a single microRNA can modulate a signaling pathway at multiple levels. In addition, we demonstrate that mammalian homologues of miR-8 antagonize the ability of Wnt signaling to block adipogenesis in marrow stromal cells. Therefore, miR-8 and its homologues appear to play an evolutionarily conserved role in regulating Wnt signaling.

Results

miR-8 Is Identified in a Genetic Screen for Antagonists of Wg Signaling.

Ectopic activation of Wg signaling in the developing eye using the GMR-Gal4 driver causes a dramatic reduction in eye size (Fig. 1B). To identify regulators of the Wg pathway, we performed a genetic screen to identify genes that, when misexpressed, suppress this small-eye phenotype (10, 11). Wg was coexpressed with random genes that were placed under the control of bidirectional Gal4-dependent (UAS) promoters by GSV transposable element insertions (12). Two GSV transposon insertions (GSV1305-2 and GSV2196), known to suppress the GMR/Wg phenotype, were located upstream of the microRNA miR-8 (data not shown). Both GSV insertions also suppressed the phenotype resulting from ectopic expression of Arm*, a stable form of Arm, in the developing eye (Fig. 1C and data not shown). To verify that the phenotype of these insertions was because of expression of miR-8 and not to expression of surrounding genes, we generated transgenic flies expressing miR-8 under the control of a Gal4-dependent promoter (UAS-miR-8). Expression of miR-8 suppressed the small-eye phenotype caused by ectopic expression of Wg or Arm* (data not shown). These data suggest that miR-8 can inhibit ectopic Wg signaling in the developing fly eye.

Fig. 1.
miR-8 identified as a potential negative regulator of Wg signaling in a genetic screen. Micrographs of adult Drosophila eyes from flies containing the eye-specific driver GMR-Gal4 alone (A) or in combination with GMR-arm* (B) or GMR-arm* and GSVA2196 ...

miR-8 Inhibits Endogenous Wg Targets in Flies.

To determine the effect of miR-8 expression on endogenous Wg signaling, we examined Wg readouts in the wing and leg imaginal discs. In the third larval instar wing imaginal disc, Wg was expressed in a stripe of cells along the dorsal-ventral boundary of the wing blade primordia (wing pouch; Fig. 2A) and activates target genes such as Distal-less (Dll, Fig. 2B) (10, 13). Expression of miR-8 along the anterior–posterior boundary of the wing pouch using decapentaplegic (dpp)-Gal4 dramatically reduced Dll expression, as visualized by a loss of Dll-lacZ reporter expression in this domain (Fig. 2D). Expression of miR-8 inhibited all other Wg targets tested, including sens and reporters for fz3, nkd, and Notum, caused notches in the adult wings [supporting information (SI) Fig. S1 and data not shown].

Fig. 2.
miR-8 inhibits several Wg readouts in vivo. (A–D) Confocal images of third instar wing imaginal disc from a WT fly (A and B) or a fly containing dpp-Gal4>UAS-miR-8 (C and D) to drive miR-8 expression along the anterior–posterior ...

In the developing leg, Wg is expressed ventrally and dpp is expressed dorsally, as visualized with dpp-lacZ (Fig. 2E). If the Wg pathway is disrupted, then dpp expression becomes derepressed in the ventral portion of the leg disc (1416). We expressed miR-8 using ptc-Gal4, a driver active in both wg and dpp expression domains. Expression of miR-8 in this domain caused derepression of dpp-lacZ, as indicated by the extension of dpp-lacZ expression into the ventral portion of the leg disc (Fig. 2F). This derepression is consistent with miR-8 expression antagonizing endogenous Wg signaling.

miR-8 Inhibits Wg Secretion by Directly Targeting wls.

One predicted target of miR-8 is wls, a transmembrane protein required for Wg secretion (17, 18). The 3′UTR of wls contains one putative miR-8–binding site, which is conserved in Drosophila pseudobscura (19) (Fig. 3A). To test whether miR-8 directly targets the 3′UTR of wls, we generated a sensor for wls by cloning the wls 3′UTR downstream of the coding region for lacZ. The wls 3′UTR sensor was suppressed by miR-8 in Kc167 cells, and mutation of the seed region (base pairs 2–8) of the putative miR-8–binding site partially blocked the ability of miR-8 to inhibit the wls sensor (Fig. 3A). Wls protein is expressed ubiquitously throughout the third larval instar wing imaginal disk, and Wls is up-regulated by the Wg pathway along the dorsal-ventral boundary (20) (Fig. 3B). Expression of miR-8 along the anterior–posterior boundary of the wing pouch using dpp-Gal4 inhibited Wls protein expression (Fig. 3D). Secretion of Wg also was decreased by miR-8 expression, as indicated by decreased extracellular Wg (Fig. 3E). Together, these data suggest that miR-8 directly targets wls and inhibits Wg signaling in part by preventing Wg secretion.

Fig. 3.
miR-8 directly targets wls, a gene required for Wg secretion. (A) Schematic of wls 3′UTR containing one putative miR-8–binding site that is conserved in Drosophila pseudobscura. Kc167 cells were transfected with 3′UTR sensors containing ...

miR-8 Can Inhibit Wg Signaling Downstream of Arm Stabilization in Cell Culture.

Expression of miR-8 suppressed the phenotype caused by expression of Arm*, a stable form of Arm, in the developing fly eye (Fig. 1). This suggests that miR-8 may target the Wg pathway downstream of Arm stability in addition to targeting wls. Expression of Arm* in Kc167 cells activates TCF-luc, a reporter gene with an enhancer that contains multimerized TCF-binding sites (21) (Fig. 4A). Activation of TCF-luc by Arm* is consistently decreased by 3- to 4-fold through coexpression of miR-8 (Fig. 4A). These data suggest that miR-8 can inhibit Wg signaling downstream of Arm stabilization both in vivo and in cell culture.

Fig. 4.
miR-8 inhibits TCF protein without affecting mRNA in cell culture and in vivo. (A) Kc167 cells were transfected with TCF-luc or UAS-luc reporter gene, pAclacZ, pAcArm*, pAcVP16-Lef, pAcGal-Arm, and pAc or pAc-miR-8, as indicated. Samples were normalized ...

To explore the mechanism of miR-8 action, we took advantage of various chimeric constructs. miR-8 expression decreased TCF-reporter activation by Arm* but not by VP16-Lef1, a fusion protein between the activation domain of VP16 and Lef1 (Fig. 4A). This finding suggests that miR-8 is not a general inhibitor of transcription. miR-8 inhibited the activation of TCF-luc by Gal-Arm, a fusion protein between the Gal4-binding domain and full-length Arm; however, miR-8 did not suppress Gal-Arm activation of a Gal-dependent reporter gene (Fig. 4A). Together, these findings suggest that miR-8 can suppress Arm activation of gene expression only when Arm is recruited to its target gene by TCF, and that miR-8 has no effect on transcription if Arm is recruited independently of TCF via the Gal4 domain. These data suggest that inhibition by miR-8 of Wg target genes depends on TCF.

miR-8 Inhibits TCF Protein Without Affecting TCF mRNA Levels.

To test for repression of TCF, we enriched for the population of cells transiently transfected with empty vector or miR-8. We found that TCF protein was decreased in cells expressing miR-8 (Fig. 4B), whereas TCF mRNA was unaffected (Fig. 4C). In contrast, we found no change in the levels of Arm protein or mRNA after miR-8 expression (Fig. 4 B and C). The data are consistent with either miR-8 targeting translation of TCF mRNA or miR-8 targeting a gene required for TCF protein stability.

To examine the effect of miR-8 on TCF protein in vivo, we expressed miR-8 in developing imaginal discs using 1J3-Gal4 (Fig. 4D). We found that TCF protein was decreased in cell lysates from wing and leg imaginal discs expressing miR-8, confirming that miR-8 inhibits TCF protein both in vivo and in cell culture.

The TCF locus, also called pangolin (pan), gives rise to multiple isoforms because of the use of alternative exons (Fig. S2A). Two 3′UTRs have been reported for TCF, one associated with full-length isoforms of TCF (82 kDa; pan-RA and others) and one associated with a short isoform (46 kDa; pan-RI). The full-length 82-kDa isoforms of TCF were down-regulated by miR-8 (Fig. 4 B and D). miR-8 is predicted to target the 3′UTR of the short isoform but not of the longer isoforms (Fig. S2B) (22). We generated sensors for both 3′UTRs and found that neither one was targeted by miR-8 (Fig. S2C). This data suggest that miR-8 does not directly target the 3′UTR of TCF mRNA transcripts.

miR-8 Directly Targets CG32767, a Positive Regulator of the Wg Pathway.

Another potential miR-8 target that we tested was CG32767, a zinc finger protein identified as a positive regulator of Wg signaling in a genome-wide RNAi screen (21, 23). The 3′UTR of CG32767 contains two putative miR-8–binding sites, one of which is conserved in Drosophila pseudobscura (24) (Fig. 5A). We generated a sensor for CG32767 by cloning a portion of the CG32767 3′UTR downstream of the coding region for lacZ. Expression of miR-8 inhibited the CG32767 sensor 3-fold in Kc167 cells (Fig. 5B). This inhibition was blocked by mutation of the base pairs of both sites predicted to bind the seed region of miR-8 (Fig. 5B). Mutation of either site alone gave only a partial rescue, suggesting that both sites are functionally important (data not shown). Knockdown of CG32767 by RNAi in Kc167 cells inhibited Wg signaling, consistent with results in other fly cell lines (data not shown) (21, 23). We do not yet know where CG32767 acts in the Wg pathway, although we found that TCF protein expression was not affected by knockdown of CG32767 (data not shown). These data suggest that CG32767 is a target of miR-8 and may contribute to the miR-8 expression phenotype seen in vivo. Together, these findings suggest that miR-8 targets the Wg pathway at multiple levels, from Wg secretion (via wls) to reception of the signal (via TCF).

Fig. 5.
miR-8 directly targets another positive regulator of the Wg pathway, CG32767. (A) Schematic of CG32767 3′UTR containing two putative miR-8–binding sites. The site depicted in black is conserved in Drosophila pseudobscura. (B) Kc167 cells ...

Mammalian miR-8 Family Members Promote Adipogenesis.

Our studies suggest that miR-8 is a potent inhibitor of the Wg signaling pathway in Drosophila melanogaster. To examine whether this regulation is conserved in vertebrates, we studied the role of mammalian homologues of miR-8 in a cell culture model of adipogenesis. Three mouse microRNAs (miR-200c, miR-429, and miR-200b) are identical to miR-8 in the seed region (residues 2–8), and two other microRNAs (miR-141 and miR-200a) are nearly identical, with a single substitution of U with C, both of which can bind G (Fig. 6A). In vertebrates, these five microRNAs are found in the genome as two clusters (miR-200c and 141, and miR-200b, 200a, and 429) located on different chromosomes.

Fig. 6.
Murine miR-8 family members promote adipogenesis and antagonize inhibition of adipogenesis by Wnt3a. (A) Alignment of Drosophila melanogaster miR-8 mature sequence with Mus musculus miR-8 family members. Seed regions are shaded in gray. (B–D) ...

We chose to study these mammalian miR-8 family members in ST2 marrow stromal cells because it has been shown that Wnt signaling potently inhibits the differentiation of ST2 cells into adipocytes (2527). Suppression of endogenous Wnt signaling in ST2 cells promotes adipogenesis, and ectopic treatment with Wnts blocks the differentiation process (data not shown) (25). We generated stable cell lines expressing the miR-200c/141 or miR-200b,a/429 cluster and induced the cells to differentiate into adipocytes. Expression of either cluster of microRNAs increased adipogenesis, as assessed by increased lipid accumulation (Fig. 6 C and D) and increased expression of FABP4, an adipocyte marker (Fig. 6E). In addition, expression of either cluster of microRNAs partially rescued the block of differentiation caused by treatment with recombinant Wnt3a (Fig. 6E). Together, these data suggest that miR-8 family members regulate adipogenesis, possibly by inhibiting Wnt signaling.

Discussion

Our studies suggest that miR-8 is a potent antagonist of Wg signaling in vivo and in cell culture. Inhibition of Wg signaling by miR-8 may be because of targeting of the pathway at multiple levels, from Wg secretion to the reception of the signal in the nucleus by TCF. Finally, we extended our studies of miR-8 across species to examine the effects of miR-8 family members in a cell culture model of mouse mesenchymal stem cell differentiation. Our studies suggest that miR-8 family members promote adipogenesis by inhibiting endogenous Wnt signaling. Negative regulation of Wnt signaling by these microRNAs is consistent with a study reporting that all five miR-8 family members are highly expressed in the epidermis of the skin but are excluded from the hair follicle, a site of Wnt activity (28). Together, our data suggest that miR-8 may play an evolutionarily conserved role in negatively regulating Wnt signaling.

We found that miR-8 inhibits TCF protein levels without affecting TCF mRNA, suggesting that TCF is a direct target of miR-8. However, the 3′UTRs of the reported TCF isoforms were not regulated by miR-8 (Fig. S2), suggesting that miR-8 may directly target TCF mRNA independently of its 3′UTR or through an indirect mechanism. An intriguing possibility is that miR-8 may directly target an unidentified gene that is required for TCF protein stability. Studies have shown that TCF/LEF transcription factor activity or subcellular localization is regulated by posttranslational modifications, such as sumoylation, phosphorylation, and acetylation; however, none of these studies have reported an effect of these modifications on overall TCF protein expression (29). Interestingly, Sox17 has been reported to negatively regulate both TCF-4 and β-catenin protein levels in human colorectal cell lines by a mechanism that appears to require the proteasome (30); however, a positive regulator of TCF protein stability has not yet been reported, and we found that knockdown of the miR-8 target CG32767 did not affect TCF protein levels (data not shown).

Another microRNA, miR-315, was recently reported to be a positive regulator of the Wg pathway (9). miR-315 was identified in a cell culture-based screen for effects on Wg dependent reporter gene activity. In contrast to our findings, that study did not identify miR-8 as a pathway regulator in its screen. These discrepant findings may be because of our use of different cell lines (Kc167 vs clone8) and overall approaches (in vivo vs cell culture-based screen). We have not tested the effects of miR-8 expression in clone8 cells, although we have noted robust inhibition of the Wg pathway in the wing imaginal disk, the original source of the cell line.

miR-8 loss of function mutant flies were reported recently (31). The authors described a subtle mutant phenotype with defective leg and wing extension and behavioral defects, in part, because of an increased expression of Atrophin. Although the miR-8 mutants do not demonstrate an obvious Wg-related phenotype, Wg signaling does play an important role in Drosophila leg and wing development (1416), and increased expression of other direct or indirect targets of miR-8 (e.g., TCF, wls, CG32767) also may contribute to the mutant phenotype in the leg and elsewhere.

The subtle phenotype of the miR-8 mutant is not surprising, given similar reports for other microRNA loss of function mutants (3). The differences in scale of the loss-of-function versus the gain-of-function phenotype that we have reported here may be, in part, because of redundancy, because all three targets that we have identified contain predicted binding sites for multiple microRNAs. Mutation of multiple microRNAs may be required to produce dramatic effects on some signaling and developmental pathways. In addition, negative and positive regulators of the Wg pathway, such as nkd and fz3, are dispensable in certain tissues, including the wing imaginal disk (32, 33). This may be indicative of redundant negative and positive regulator activities in these tissues. Redundancy may protect the Wg pathway in certain tissues from aberrations because of alterations in regulator gene expression. Overall, the studies of miR-8 suggest that the role of miR-8 is not as a developmental switch, but instead miR-8 may act as a modulator of multiple pathways in vivo by precisely tuning target gene expression in concert with other microRNAs and genes.

Materials and Methods

Drosophila Genetics.

Insertions of a bidirectional EP element, Gene search vector (GSV) (12), were screened for the ability to suppress GMR-Gal4>UAS-wg and GMR-Gal4>GMR-arm*, as described (10, 11) (see SI Text). Two GSV elements were mapped by inverse PCR to a region upstream of miR-8 (GSVA1305–2 and GSVA2196). Both GSV lines gave similar phenotypes in all assays. dpp-Gal4, ptc-Gal4, Dll-lacZ, dpp-lacZ (BS 3.0), and 1J3-Gal4 were obtained from the Bloomington Stock Center.

UAS-miR-8 lines were generated by cloning a 500-bp genomic fragment containing the miR-8 hairpin into the pUAST vector (BestGene) (34). Ten independent lines were obtained, all of which gave phenotypes similar to those shown in Figs. 113. Two of the lines, 1F-20 (second chromosome insertion) and 1F-34 (third chromosome insertion), were used for experiments described in Figs. 2 and and33.

Plasmids.

All expression plasmids used for transfection of Kc167 cells were expressed under control of the Actin promoter using pAc5.1/V5-His-A (Invitrogen). pAc-miR-8 was generated by cloning a 500-bp genomic fragment containing the miR-8 hairpin. pAcArm* expresses a stabilized form of Arm as described (35). VP16-Lef1 is a fusion protein of the activation domain of VP16 with Lef1 (36). A fusion protein of the DNA-binding domain of Gal4 and full-length Arm was reported (10). pMSCV-miR-200c/141 and pMSCV-miR-200b,a/429 were generated by cloning into pMSCV (Clontech) PCR-amplified genomic regions containing the clustered hairpins of miR-200c and miR-141 or miR-200b, miR-200a, and miR-429 from mouse genomic DNA.

The TCF-responsive reporter gene, TCF-luc (12XdTCF) (21), was obtained from N. Perrimon (Harvard Medical School, Boston). The Gal4-responsive reporter gene, UAS-luc, was reported (10). The CG32767 3′UTR reporter gene was generated by cloning a stop codon, followed by a 500-bp fragment of the CG32767 3′UTR containing the two predicted miR-8–binding sites downstream of the lacZ coding region in the pAclacZ vector (Invitrogen). The wls 3′UTR reporter gene was generated by cloning the entire 3′UTR of wls downstream of lacZ in pAclacZ. Mutant 3′UTR reporter genes contain CAGTATT to ACTGCGG point mutations in the predicted seed matches. Cloning details are available on request.

Whole-Mount Staining and Microscopy.

Immunostaining was performed as described (37). Anti-Wg and anti-lacZ were obtained from the Developmental Studies Hybridoma Bank and Abcam, respectively, and anti-Wls was obtained from K. Basler (University of Zurich, Zurich) (20). Immunostaining for extracellular Wg was performed as described (38). Fluorophore-conjugated secondary antibodies were obtained from Jackson ImmunoResearch and Molecular Probes. All confocal fluorescent pictures with Z-stack projection were obtained by using Leica apparatus. All adult fly head micrographs were taken with a Leica MZ APO light microscope.

Cell Culture.

Drosophila Kc167 cells were routinely cultured as described (10). Mouse ST2 marrow-derived stromal cells were cultured and induced to differentiate into adipocytes as described (27). To visualize lipid accumulation, cells were stained with Oil Red-O 6 days after induction of differentiation (26). miR-200c/141 or miR-200b, a/429, or empty pMSCV vector were stably expressed in ST2 cells by retroviral infection, followed by antibiotic selection, as described (27).

Transient transfections of Kc167 cells and luciferase and β-galactosidase assays were carried out as described (39). Transfection details are available on request. All experiments using cell cultures were repeated at least three times, with two or more replicates in each independent experiment. All error bars represent a standard deviation of at least two replicates in a representative experiment.

Immunoblotting and Quantitative RT-PCR.

Purification of transfected Kc167 cells was accomplished as described (39). Purified cells were either lysed for immunoblotting or isolation of total RNA. To determine TCF protein expression in wing and leg imaginal discs, control 1J3-Gal4 larvae or 1J3-Gal4>UAS-miR-8 larvae were raised at 18°C and switched to 29°C 24 h before dissection. Antisera raised against the N terminus of TCF (10), Arm antibody (Developmental Studies Hybridoma Bank), and Tubulin antibody (Sigma Aldrich) were used for immunoblotting. For quantitative RT- PCR, RNA was reversed-transcribed with Stratascript reverse transcriptase (Stratagene), followed by qualitative PCR analysis for TCF, arm, and Tubulin56D transcripts, as described (10).

Stable ST2 cell lines were treated with recombinant mouse Wnt3a (R&D Systems) for the first 4 d of adipocyte induction. Cells were lysed, and anti-FABP4 (R&D Systems) was used for immunoblotting 6 d after the start of induction.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank members of the Cadigan Lab, University of Michigan, for helpful discussions. This work was supported by National Institutes of Health Grant R01GM082994 (to K.M.C.) and a National Institutes of Health Individual NRSA Postdoctoral Fellowship (to J.A.K.).

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0807763105/DCSupplemental.

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