Lkb1 is required for TGFbeta-mediated myofibroblast differentiation.

Inactivating mutations of the tumor-suppressor kinase gene LKB1 underlie Peutz-Jeghers syndrome (PJS), which is characterized by gastrointestinal hamartomatous polyps with a prominent smooth-muscle and stromal component. Recently, it was noted that PJS-type polyps develop in mice in which Lkb1 deletion is restricted to SM22-expressing mesenchymal cells. Here, we investigated the stromal functions of Lkb1, which possibly underlie tumor suppression. Ablation of Lkb1 in primary mouse embryo fibroblasts (MEFs) leads to attenuated Smad activation and TGFβ-dependent transcription. Also, myofibroblast differentiation of Lkb1–/– MEFs is defective, resulting in a markedly decreased formation of α-smooth muscle actin (SMA)-positive stress fibers and reduced contractility. The myofibroblast differentiation defect was not associated with altered serum response factor (SRF) activity and was rescued by exogenous TGFβ, indicating that inactivation of Lkb1 leads to defects in myofibroblast differentiation through attenuated TGFβ signaling. These results suggest that tumorigenesis by Lkb1-deficient SM22-positive cells involves defective myogenic differentiation.


Introduction
Peutz-Jeghers syndrome (PJS) is caused by inactivating mutations of the LKB1 (STK11) serine/threonine kinase gene, and is characterized by gastrointestinal polyposis and an increased risk of cancer (Hemminki et al., 1998). PJS polyps are hamartomas, consisting of differentiated epithelia and a prominent stromal compartment with a characteristic smooth-muscle core. Recently, it was demonstrated that stromal mutations of Lkb1 are sufficient for epithelial proliferation and polyposis in a PJS mouse model by conditional deletion of Lkb1 in SM22 (also known as Tagln)expressing mesenchymal cells .
The identified tumor-suppressive function of Lkb1 in the Lkb1 lox/+ ;Tagln Cre/+ mice focuses interest towards changes in SM22-expressing cells following Lkb1 deletion, especially because these changes probably underlie polyposis also in PJS patients . Immunohistochemical analysis of mouse and human polyps revealed a prominent increase in stromal αsmooth muscle actin (SMA)-expressing cells, which did not display the smooth-muscle cell (SMC) marker desmin. This phenotype is characteristic of myofibroblasts that were originally identified in the granulation tissue of wounds as highly contractile fibroblastic cells with SMC features Tomasek et al., 2002). Importantly, characteristics include the expression of SM22 in several tissues (Chiavegato et al., 1999;Faggin et al., 1999). Thus, the additional myofibroblasts in the conditional Lkb1 polyposis model could originate from the small number of myofibroblasts that are normally present, which proliferate following Lkb1 deletion; a similar proliferation of pericryptal myofibroblasts has been suggested as a possible mechanism for the increased number of myofibroblasts in other polyposis models (Powell et al., 2005).
Shared markers, such as SMA and SM22, and the high plasticity of fibroblasts, myofibroblasts and SMCs in, for example, vascular injury have raised the concept that these cell types represent a continuous spectrum of differentiation stages Tomasek et al., 2002;Zalewski et al., 2002). Furthermore, dedifferentiation of SMCs to myofibroblasts has been noted, e.g. following vascular injury (Rajkumar et al., 2005) and in severe lesions of the coronary artery (Hao et al., 2006). Thus, an alternative model for the increased number of myofibroblasts in Lkb1 lox/+ ;Tagln Cre/+ polyps is dedifferentiation of the more abundant SMCs following Lkb1 deletion. At present, Lkb1 has not been directly implicated in myogenic differentiation despite conditional mutagenesis studies in skeletal (Sakamoto et al., 2005a) and cardiac (Sakamoto et al., 2005b) muscle. However, the importance of Lkb1 in neuronal polarization, migration and differentiation (Asada et al., 2007;Barnes et al., 2007;Shelly et al., 2007) suggests a celltype-specific requirement for Lkb1 in differentiation.
Deficient TGFβ signaling from Lkb1-deficient stroma to epithelia was identified as one possible mechanism for epithelial hyperproliferation and tumorigenesis , but it is yet unknown how deletion of LKB1 can induce the formation of myofibroblasts in polyposis. Interestingly, TGFβ is a crucial factor in both myofibroblast (Desmouliere et al., 1993;Hinz et al., 2001b) and SMC (Grainger et al., 1998;Roelofs, 1998;Qiu et al., 2006) differentiation.
When fibroblasts that are isolated from tissues are grown on standard tissue-culture plates, focal-adhesion attachment to the rigid Inactivating mutations of the tumor-suppressor kinase gene LKB1 underlie Peutz-Jeghers syndrome (PJS), which is characterized by gastrointestinal hamartomatous polyps with a prominent smooth-muscle and stromal component. Recently, it was noted that PJS-type polyps develop in mice in which Lkb1 deletion is restricted to SM22-expressing mesenchymal cells. Here, we investigated the stromal functions of Lkb1, which possibly underlie tumor suppression. Ablation of Lkb1 in primary mouse embryo fibroblasts (MEFs) leads to attenuated Smad activation and TGFβ-dependent transcription. Also, myofibroblast differentiation of Lkb1 -/-MEFs is defective, resulting in a markedly decreased formation of α-smooth muscle actin (SMA)-positive stress fibers and reduced contractility. The myofibroblast differentiation defect was not associated with altered serum response factor (SRF) activity and was rescued by exogenous TGFβ, indicating that inactivation of Lkb1 leads to defects in myofibroblast differentiation through attenuated TGFβ signaling. These results suggest that tumorigenesis by Lkb1-deficient SM22-positive cells involves defective myogenic differentiation.
non-deformable plastic leads to tension and subsequent differentiation of cells to myofibroblasts Wipff et al., 2007). Here, we used this myofibroblast-differentiation model using primary mouse embryonic fibroblasts (MEFs) with a loxed allele of Lkb1 to investigate the involvement of Lkb1 in TGFβ signaling and myofibroblast differentiation.

Lkb1 regulates mRNA levels of TGFβ1
Supernatants from Lkb1 -/-MEF culture were previously noted to contain lower levels of TGFβ , which could reflect defective production (Yue and Mulder, 2000), defective secretion (Miyazono et al., 1991) or defective extracellular processing (Annes et al., 2003) of TGFβ. To investigate possible mechanisms behind the decreased levels of TGFβ in culture supernatants, we used primary MEFs with a loxed allele of Lkb1 (Bardeesy et al., 2002), enabling conditional deletion of Lkb1 following the introduction of Cre recombinase using a recombinant adenovirus (AdCre) (Anton and Graham, 1995).
To determine possible changes in TGFβ production, mRNA levels of TGFβ1 were determined from Lkb1 lox/-MEFs infected with recombinant adenovirus containing lacZ (AdLacZ) (referred herein as control; see Materials and Methods) and AdCre-infected Lkb1 lox/-MEFs (Lkb1 -/-) grown in the presence of 10% fetal calf serum (FCS). Real-time PCR analysis of GAPDH-normalized mRNA levels (see Materials and Methods) demonstrated a 47% reduction of mRNA levels of TGFβ1 in Lkb1 -/-MEFs, suggesting that the previously noted comparable changes in supernatants  were due to Lkb1-mediated intracellular changes that were reflected in altered mRNA levels.
To validate these results, a second genetic approach using siRNA-mediated knockdown of Lkb1 in wild-type MEFs was used.
Attenuated activity of the Smad2 and Smad3 pathways in Lkb1 -/cells To further investigate Smad2 activation in Lkb1 -/-MEFs, levels of active Smad2 phosphorylated on serines 465 and 467 (Smad2-P) were analyzed by western blotting. Smad2-P levels in untreated samples were robust in control MEFs, but significantly lower in Lkb1 -/-MEFs (Fig. 1B). The addition of high levels (1 ng/ml) of exogenous TGFβ increased Smad2-P levels both in control and Lkb1 -/-MEFs (Fig. 1B). Attenuated Smad activation in Lkb1 -/-MEFs was also suggested by immunofluorescence analysis. In Lkb1 -/-MEFs, Smad2 and Smad3 were mostly detected in a cytoplasmic localization, which changed to a predominant nuclear staining following the addition of TGFβ (1 ng/ml). This staining was comparable to control MEFs even in the absence of exogenous TGFβ (Fig. 1C).
Although TGFβ signaling is attenuated at the R-Smad and transcriptional level in Lkb1-deficient MEFs, the results above did not allow determination of the level of regulation due to autoinduction by TGFβ1. To now differentiate whether defects following deletion of Lkb1 were primarily at the level of TGFβ production or in intracellular TGFβ signaling, the inducibility of the Smad3 reporter (CAGA) 12 -luc to exogenously added TGFβ was assessed. The addition of a saturating concentration (1 ng/ml; based on saturation curves) of active TGFβ to the culture medium resulted in the induction of (CAGA) 12 -luc both in control and Lkb1 -/-MEFs, but activity was still significantly lower in Lkb1 -/-MEFs (37% of control) (Fig. 1D, vector + TGFβ). The result suggested a defect between the formation of active TGFβ and target-gene induction. To further dissect the level at which the defect occurs, (CAGA) 12 -luc activity was compared in cells transfected with a constitutively active (ca) TGFβ type-I receptor (TGFβRI) (Nakao et al., 1997); again, (CAGA) 12 -luc activity was significantly lower in Lkb1 -/-MEFs (22% of controls) (Fig. 1D, caALK5). These results suggest that the defects noted in Lkb1 -/-MEFs are primarily due to downregulation of intracellular TGFβ signaling resulting in reduced TGFβ levels in culture supernatants. Lkb1 in myofibroblast differentiation in the MEF cultures because a significant fraction of MEFs spontaneously differentiate into myofibroblasts in a rigid environment (Chen et al., 2004;Greenberg et al., 2006;Sousa et al., 2007), represented, for example, by a normal-tissue-culture plate. Here, this spontaneous differentiation of MEFs was found to be dependent on the endogenously produced TGFβ, because addition of a TGFβRI inhibitor, SB431542 (Inman et al., 2002), led to significantly decreased expression of SMA, the expression of which was otherwise noted in 75% of the cells thereby defined as myofibroblasts ( Fig. 2A).
Myofibroblast differentiation, as well as TGFβ signaling, has been associated with various effects on proliferation depending on the experimental conditions (Desmouliere et al., 1992;Grotendorst et al., 2004;Ng et al., 2005;Sorrentino and Bandyopadhyay, 1989;White et al., 2006) or concentration of TGFβ (Battegay et al., 1990). However, the noted differences in SMA and SM22 levels and in TGFβ signaling of Lkb1 -/-MEFs were not associated with a significant change in proliferation rates, because comparable fractions of BrdU-positive cells following a 6-hour pulse of BrdU were observed (supplementary material Fig. S1), which is consistent with previous observations on early-passage Lkb1 -/-MEFs (Bardeesy et al., 2002).

Lkb1 MEFs have decreased contractility
Another important feature of myofibroblasts is the development of contractile stress fibers that define the 'proto-myofibroblasts' that precede myofibroblasts expressing SMA Tomasek et al., 2002). To test whether Lkb1 -/-MEFs also exhibit defective stress-fiber formation, F-actin was visualized with fluorescent phalloidin. In contrast to the prominent stress fibers observed in control MEFs, contractile bundles were lacking in Lkb1 -/-MEFs, in which F-actin was restricted to the cell periphery ( Fig. 2D, phalloidin). Concomitantly with the reduction of stress fibers and reorganization of F-actin in Lkb1 -/-MEFs, focal Fold induction is indicated relative to control samples and all bars represent averages of three experiments performed with independently isolated MEFs using triplicate samples, except for the (CAGA) 12 -luc Lkb1 lox/+ and (CAGA) 12 -luc Lkb1 lox/lox bars, which represent averages from ten and five experiments, respectively. Right panel shows similar analysis with MEFs transfected with non-targeting (control) or Lkb1-targeting (siLkb1) siRNAs and (CAGA) 12 -luc. Bars represent averages from two experiments using four to ten samples per experiment. Error bars indicate s.d.; **P<0.01, ***P<0.001. (B) Western blotting analysis of Lkb1, activated Smad2 (Smad2-P, shown as p-Smad2) and total Smad2 from serumstarved (0.2% FCS) untreated (-) or TGFβ-treated (+; 1 ng/ml) control (AdLacZ-infected Lkb1 lox/-MEFs) or Lkb1 -/-(AdCre infected Lkb1 lox/-MEFs) MEFs. A background band in the Smad2-P blot is indicated with an asterisk. (C) Immunofluorescence staining using an antibody recognizing Smad2 and Smad3 of serum-starved (0% FCS, 22 hours) and subsequently untreated (-) or TGFβ-treated (1 ng/ml) control and Lkb1 -/-MEFs. The apparently lower total intensity of signal might be due to more diffuse localization of Smad2 and Smad3 in the cytoplasm of Lkb1 -/-MEFs compared with controls because no difference in total levels were noted in western blotting (see Fig. 1B). Nuclei are visualized with Hoechst. A single-cell magnification is shown in the inset. (D) (CAGA) 12 -luc activity in control and Lkb1 -/-MEFs transfected with constitutively active ALK5 (ca-ALK5) or vector control and treated with added TGFβ as indicated. The value of relative (CAGA) 12 -luc activity represents the ratio of (CAGA) 12 -luc to Renilla-luciferase-thymidine-kinase (RL-TK), with the average from a representative experiment out of three using triplicate samples shown; error bars indicate s.d.
The dramatic effect of Lkb1 depletion on focal-adhesion morphology and distribution provoked the question: is FAK activation altered in Lkb1 -/-MEFs? To assess FAK activation upon cell adhesion, control and mutant MEFs were seeded either on adhesive fibronectin-coated plates or on non-adhesive bovine serum albumin (BSA)-coated plates. Analysis of FAK activity 50 minutes after plating demonstrated comparable FAK activity specifically on the fibronectincoated plates [ Fig. 3A, Tyr-P (p-Tyr), FN]. Because fibronectin coating did not alter the level of SMA expression in control or Lkb1 -/-MEFs (data not shown), the results suggest that initial cell adhesion and FAK activation are normal in the absence of Lkb1.

Lkb1 -/-MEFs exhibit normal SRF activity
TGFβ signaling has been suggested to promote myofibroblast differentiation via CArG-box-mediated transcription through increasing SRF levels (Jeon et al., 2006;Qiu et al., 2003) and inducing nuclear localization of the SRF cofactor MRTF-A (also known as MKL1 and MAL) (Wang et al., 2002) in a RhoA-dependent manner Miralles et al., 2003). To monitor MRTF-A and Journal of Cell Science 121 (21)  (Cen et al., 2003;Murai and Treisman, 2002) was transfected into control and Lkb1 -/-MEFs, followed by a serum-starvation/restimulation experiment. SRE-luc activity was comparable between control and Lkb1 -/-MEFs under starved conditions (Fig. 3B, 0% FCS), in which both TGFβ signaling and myofibroblast markers are deregulated in Lkb1 -/-MEFs. Following serum stimulation, induction of SRE-luc was maximal at 3 hours and decreased at 15 hours in both control and mutant MEFs, with the small and opposite differences observed between the controls and mutants at the two time points considered to not be likely to represent meaningful differences (Fig. 3B, 20% FCS). The addition of TGFβ (see Materials and Methods) to the starved sample had only a modest and comparable effect on SRE-luc activity in control and Lkb1 -/-MEFs at 3 and 15 hours (Fig. 3B, TGFβ). Consistent with unaltered SRE-luc activity, immunofluorescence analysis of MRTF-A indicated comparable subcellular distribution in control and Lkb1 -/-MEFs (data not shown). Unaltered SRF activity was also supported by the fact that PKB (AKT) activity (as assessed by Ser473 phosphorylation) was similar between control and Lkb1 -/-MEFs [ Fig.  3C, PKB-P (p-PKB)], because PKB has been suggested to regulate TGFβ-dependent SMA expression by inducing SRF binding to SREs (Lien et al., 2006). Also, independent evidence for unaltered RhoA activity was provided by the fact that there were comparable levels of moesin that was phosphorylated on Thr558 [ Fig. 3C, moesin-P (p-Moesin)], a site that is phosphorylated by the RhoA-activated Rho kinase (Matsui et al., 1998;Oshiro et al., 1998). These results suggest that the changes in expression of SMA and SM22 in Lkb1 -/-MEFs are not mediated through alterations of RhoA-SRF signaling.

Unaltered myofibroblast differentiation in
The kinase AMPK has been suggested to regulate the contractile machinery in parallel with RhoA via phosphorylation of the regulatory myosin light chain (MLC) (Lee et al., 2007), and AMPK requires Lkb1 phosphorylation for activation (Hawley et al., 2003). To study whether defects in myofibroblast differentiation were due to decreased activation of AMPK after depletion of Lkb1, we stained control and AMPKα1 -/-;AMPKα2 -/-MEFs (see Materials and Methods) for SMA. AMPKα1 -/-;AMPKα2 -/-MEFs exhibited no alterations in the levels of SMA or SM22 (supplementary material Fig. S2) in conditions in which Lkb1 -/-MEFs demonstrated a significant decrease in SMA expression ( Fig. 2A). Thus, whereas stimulation of AMPK by exogenously added AMP-mimic AICAR appears to interfere with myofibroblast differentiation (Mishra et al., 2008), lack of AMPK does not significantly alter myofibroblast differentiation in unstimulated conditions, and AMPK does not appear to be involved in the observed loss of differentiation in Lkb1 -/-MEFs.

Exogenous TGFβ is sufficient to restore SMA and SM22 expression in Lkb1 -/-MEFs
The ability of the ALK inhibitor SB431542 to downregulate SMA in primary MEFs ( Fig. 2A) implied that attenuated TGFβ signaling was responsible for decreased myofibroblast marker expression in Lkb1 -/-MEFs. To investigate this directly, Lkb1 -/-MEFs were treated with 1 ng/ml exogenous TGFβ for 45-48 hours. Importantly, the added TGFβ significantly increased the number of SMA-positive Lkb1 -/-MEFs (Fig. 4A, compare Lkb1 -/and Lkb1 -/-+ TGFβ), in addition to significantly increasing total levels of both SMA and SM22 (Fig. 4B) as shown by western blotting analysis (CDK7 levels are shown as a loading control).
TGFβ treatment was also able to rescue the contractile phenotype in Lkb1 -/-MEFs by inducing the relocalization and maturation of focal adhesions from the cell edge towards the cell center and by increasing the number of central stress fibers (Fig. 4C). Furthermore, the addition of exogenous TGFβ increased the percentage of Lkb1 -/-MEFs that were capable of deforming 10 kPa silicone substrates from 15.9% to control levels (50.7%; s.d.=15.6%, P=0.040). Thus, defective contractility of Lkb1-deficient MEFs is mediated by downregulated TGFβ signaling.
Decreased TGFβ-dependent promoter activity of myofibroblast marker genes in Lkb1 -/-MEFs TGFβ can regulate myofibroblast differentiation through a variety of different transcriptional and non-transcriptional mechanisms, among which Smad signaling appears to be the most prominent (Gu et al., 2007;Li et al., 2002). To investigate the mechanism in our model, MEFs were transfected with a SMA-luciferase reporter construct (SMA-luc) (Yoshida et al., 2003) and were treated with the TGFβRI inhibitor SB431542. After a 20-hour inhibition, SMAluc activity was reduced to 58% of control levels (Fig. 4D, SB431542, P=4.45ϫ10 -5 ). Also, transfection of MEFs with Smad7, an inhibitor of R-Smads, reduced SMA-luc reporter activity (72%, Fig. 4D, Smad7, P=0.019), indicating that endogenous Smad signaling contributes on the transcriptional level to SMA expression, and thus to myofibroblast differentiation, in primary MEF cultures.

Lkb1 modulates TGFβ signaling
The studies presented here demonstrate that Lkb1 is required for myofibroblast differentiation by modulating TGFβ signaling. This was noted through the observation of reduced Smad2-and Smad3dependent reporter activities, reduced Smad2-P levels, and the inability of exogenous TGFβ or constitutively active ALK5 to fully activate TGFβ signaling in Lkb1 -/-MEFs. Taken together, the results suggest a modulatory role for Lkb1 downstream of TGFβRI and upstream of TGFβ-dependent transcription, and provide a mechanism for the noted decreased secretion of active TGFβ by Lkb1 -/-MEFs . Previously, Lkb1 has been reported to indirectly interact with Smad4 through LIP1, a protein that has been identified as a two-hybrid interaction partner of Lkb1 (Smith et al., 2001). However, our study provides genetic evidence for normal Smad4, Smad6 and Smad7 signaling in the absence of Lkb1 based on normal BMP signaling activity (Miyazawa et al., 2002). Thus, other reported interactions (Al-Hakim et al., 2005;Brajenovic et al., 2004;Lizcano et al., 2004) are considered as more probable candidates in mediating the activity of Lkb1 on the TGFβ signaling pathway but, at this point, indirect mechanisms cannot be ruled out.
The involvement of Lkb1 in regulating TGFβ signaling suggest a novel function for Lkb1, which is likely to be independent of the previously reported LKB1-AMPK signaling (Hawley et al., 2003). Considering the multiple functions of Lkb1, it is interesting to note that a small but significant part of the transcriptomic response following loss of Lkb1 appears to be mediated via TGFβ signaling (K.V., Jianmin Wu and T.P.M., unpublished). Regulation of TGFβ signaling by Lkb1 is not restricted to fibroblasts, because deletion of Lkb1 in endothelial cells results in decreased Smad activation (as measured by Smad2-P and mRNA levels of TGFβ1 and PAI1) Journal of Cell Science 121 (21) and defective embryonal angiogenesis in vivo, which are rescued by exogenous TGFβ ex vivo (Londesborough et al., 2008).

Role of LKB1 in myofibroblast and SMC differentiation
Here, Lkb1 -/-MEFs lack the characteristics of both the first phase of proto-myofibroblast differentiation, characterized by stress-fiber formation, and further development into the differentiated myofibroblast, characterized by SMA expression and increased contractility. Platelet-derived growth factor (PDGF) has previously been suggested to stimulate early myofibroblast differentiation (Bostrom et al., 1996;Rubbia-Brandt et al., 1991). However, this pathway appears to be unaffected upon deletion of Lkb1, as was deduced from the observation of normal SRE activities in Lkb1 -/-MEFs, and similar growth stimulatory effects of PDGFβ on control and Lkb1 -/-MEFs (data not shown). In support of these findings, a previous study demonstrated unaltered ERK activity in Lkb1deficient MEFs (Shaw et al., 2004). Instead, the essential role of R-Smads in SMA-luc reporter activity in primary MEFs, the defective Smad2 and Smad3 activation and TGFβ signaling in Lkb1 -/-MEFs, and the ability of exogenous TGFβ to rescue, in Lkb1 -/-MEFs, (1) stress-fiber formation, (2) SMA and SM22 expression, (3) reporter activities and (4) force production suggest TGFβ signaling and TGFβ control elements (TCE) (Adam et al., 2000;Hautmann et al., 1997;Liu et al., 2003), and/or Smad-binding elements (SBE) (Hu et al., 2003;Qiu et al., 2005), as the effectors of Lkb1-dependent regulation of myofibroblast differentiation. These results support the previous report arguing sufficiency of TGFβ in inducing both the early and later phases of myofibroblast differentiation (Vaughan, 2000) as opposed to a role for PDGF in the early phases. In this regard, it is interesting to note that LKB1 overexpression was sufficient to increase expression of both PAI1 and SM22 in HeLa cells (Lin-Marq et al., 2005), indicating that LKB1 expression alone is sufficient to trigger expression of a TGFβ target gene (PAI1) and a myofibroblast marker gene (SM22) in epithelial cells directly.
Polyps in both Lkb1 +/mice (Bardeesy et al., 2002;Jishage et al., 2002;Miyoshi et al., 2002;Rossi et al., 2002) and mice in which Lkb1 deletion is restricted to SM22-expressing cells  contain an increased ratio of myofibroblast-like cells to SMCs in comparison to normal stomach tissue . Enrichment of myofibroblasts might be caused by their increased proliferation or dedifferentiation of SMCs. Primary myofibroblasts, the differentiation of which is controlled by the same elements controlling SMC differentiation (Hautmann et al., 1999), provide a valuable tool for in vitro studies to dissect mechanisms leading to polyp development in vivo. Whereas loss of Lkb1 and subsequent dedifferentiation of myofibroblasts did not noticeably affect proliferation of MEFs in culture, responses of various SMClike lineages to dedifferentiation in vivo can include changes also in the proliferation of dedifferentiated cells (Han et al., 2008;Kocher et al., 1991). Interestingly, the observation of haploinsufficiency of Lkb1 in myofibroblast differentiation as measured by SMA reporter activity (Fig. 4D) resembles the situation in vivo in heterozygote PJS patients and mice, in which haploinsufficiency of Lkb1 leads to polyposis (Hernan et al., 2004;Jishage et al., 2002;Rossi et al., 2002). Thus, the role of Lkb1 in the differentiation of SM22-positive cells that was investigated here suggests that aberrant differentiation of the SM22-positive cell lineage in mice underlies polyposis.
Previously, Lkb1 has been shown to be required for differentiation of neurites in vitro and in vivo (Asada et al., 2007;Barnes et al., 2007;Shelly et al., 2007), and deletion of Lkb1 has been suggested to influence lineage choice of a common lung-cancer progenitor cell (Ji et al., 2007). By contrast, ablation of Lkb1 in vivo in keratinocytes (Gurumurthy et al., 2008), liver (Shaw et al., 2005), skeletal muscles (Sakamoto et al., 2005a) or heart (Sakamoto et al., 2005b) did not provide evidence for dedifferentiation, which is in correlation with our observation of normal myoblast differentiation of Lkb1 -/-MEFs upon adenoviral-mediated MyoD (also known as MyoD1) expression (M.T. and T.P.M., unpublished). Thus, the requirement of Lkb1 for myofibroblast differentiation demonstrates that a highly cell-type-specific function is involved in tumor suppression by Lkb1.

Quantitative real-time PCR
RNAs of duplicate samples were isolated using the RNeasy isolation kit (Qiagen) according to the manufacturer's protocol. Total RNA was converted using Taqman reverse-transcription reagents (Applied Biosystems). The ABI Prism 7500 was used with Power SYBR Green PCR Master Mix (Applied Biosystems). Relative mRNA amounts were assayed by using 7500 Fast Real-Time PCR System software. Values were first compared with those obtained with GAPDH and then normalized to mRNAs isolated from control cells. The primers used were: TGFβ1 forward, 5Ј-ACTTTAGGAAGGACCTGGGTTG-3Ј; TGFβ1 reverse, 5Ј-GTAGT AG ACGA -TGGGCAGTGG-3Ј; GAPDH forward, 5Ј-AAGGTCGGAG TCA ACGGATT-3Ј; GAPDH reverse, 5Ј-TTGATGACAAGCTTCCCGTT-3Ј.

Transfection and reporter-gene assay
Transcription of luciferase in the (CAGA) 12 -luc reporter construct is controlled by 12 repeats of the consensus binding site (CAGA) of a complex consisting of Smad3 and Smad4 (Dennler et al., 1998), in ARE-luc by two repeats of ARE that is specific for Smad2-and Fast-1-dependent TGFβ-induced transcription (Yakymovych et al., 2001), in BRE-luc by SBEs from the ID1 gene that are specific for Smad1-, Smad5and Smad8-dependent BMP signaling (Korchynskyi and ten Dijke, 2002), in SMAluc by the promoter fragment (-2560 to +2784) of the SMA gene (Yoshida et al., 2003), in SM22-luc by the promoter fragment (-450 to +88) of the SM22 gene (Yoshida et al., 2003) and in SRE-luc by tandem repeats of SRE (Clontech's Mercury Pathway Profiling System).
Luciferase activity was measured at 24-48 hours after reporter-construct transfection with the Dual luciferase reporter assay system (Promega) according to the manufacturer's recommendations. The activity of firefly luciferase was normalized to that of Renilla luciferase from the co-transfected RL-TK plasmid, and fold induction was calculated with respect to the DMSO-treated (Fig. 4D) or control infected/ transfected and non-treated samples.

Contractility assay
Untreated and TGFβ-treated (1 ng/ml, 48 hours) MEFs were plated on wrinkling silicone elastomer substrates with a stiffness of 10 kPa (provided by B.H.). At this stiffness, formation of wrinkles in the substrate surface is restricted to highly contractile myofibroblasts (Hinz et al., 2001a;Hinz et al., 2002). Following 12 hours of culture, cells were fixed with 3% PFA and 300 cells/culture were scored for the ability to form wrinkles.

Statistical analysis
Statistical analyses for significance of results were performed by two-tailed Student's t-test assuming unequal variance.
We are grateful to Peter ten Dijke for Smad2-P antibody, (CAGA) 12luc, ARE-luc, BRE-luc and Fast-1 expression vectors; to Aristidis Moustakas for caALK5 and Smad7 expression vectors; to Gary Owens for SMA-luc and SM22-luc reporters; to Yuan Zhu and Luis Parada for AdCre and AdLacZ constructs; to Daniel Rifkin for MLEC cells; to Nabeel Bardeesy