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Home , GLI1, GLI2, GLI3, Homeobox, MSX1, Sonic hedgehog

signaling activates a positive feedback mechanism involving insulin-like growth factors to induce osteoblast differentiation

Yu Shia, Jianquan Chena,1, Courtney M. Karnera, and Fanxin Longa,b,c,2

Departments of aOrthopaedic Surgery, bMedicine, and cDevelopmental Biology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved February 27, 2015 (received for review February 10, 2015) Hedgehog (Hh) signaling is essential for osteoblast differentiation Insulin-like growth factors (Igfs) have been implicated in bone in the endochondral during embryogenesis. However, the formation (18). Igf1 and Igf2 both signal through Igf1 molecular mechanism underlying the osteoblastogenic role of Hh is (Igf1r) to activate PI3K/Akt/mTORC1 and Ras/Raf/Erk path- not completely understood. Here, we report that Hh markedly ways (19). Igf signaling also activates mTORC2 via a mechanism induces the expression of insulin-like growth factor 2 (Igf2) that that is not well understood, but requires PI3K and inversely activates the mTORC2-Akt signaling cascade during osteoblast correlates with mTORC1 activity (20, 21). The activity of Igfs is differentiation. Igf2-Akt signaling, in turn, stabilizes full-length modulated by the Igf-binding Igfbp1–6incomplexways Gli2 through Serine 230, thus enhancing the output of transcrip- involving both inhibitory and stimulatory effects (22). Igf1 knock- tional activation by Hh. Importantly, genetic deletion of the Igf out mice exhibit defects in bone formation, whereas overexpression signaling receptor Igf1r specifically in Hh-responding cells dimin- of Igf1 in osteoblasts enhanced their activity (23, 24). Similarly, ishes bone formation in the mouse embryo. Thus, Hh engages Igf deletion of Igf1r in the osteoblast lineage impaired bone formation signaling in a positive feedback mechanism to activate the osteo- (25, 26). Mechanistically, Igf signaling has been proposed to genic program. stimulate Runx2 activity downstream of either Akt or Erk (27, 28). We have shown that Igf and Ihh signaling control cartilage de- Hedgehog | Igf | Gli2 | Akt | osteoblast velopment largely independently, but it is not known whether the two signals intersect during osteoblast differentiation (29). he Hedgehog (Hh) proteins are secreted signaling molecules Here, we discover that Hh-Gli2 signaling markedly increases Tthat regulate many developmental processes in Igf2 mRNA and activates the Igf-mTORC2-Akt signaling cas- ranging from to (1, 2). In mammals, three cade. Akt, in turn, stabilizes full-length Gli2 via direct phosphor- members of the Hh family, i.e., Indian hedgehog (Ihh), Sonic ylation at Ser230, thus augmenting Hh-induced transcriptional hedgehog (Shh), and Desert hedgehog (Dhh), are expressed in activation. These findings identify a Hh-Igf positive feedback mechanism that operates during osteoblast differentiation. diverse tissues. Upon binding to the cell-surface receptor Patched 1 (Ptch1), Hh triggers the relocation of the seven-pass trans- Results membrane (Smo) to the primary cilium, Hh Induces Igf Signaling During Osteoblast Differentiation. To in- resulting in activation of the downstream events (3). Hh signaling vestigate the molecular mechanism responsible for Hh-induced can also be activated artificially by small molecules such as pur- osteoblast differentiation, we first established an in vitro differ- morphamine that directly targets Smo (4, 5). A major output of entiation system where the murine bone marrow stromal-derived Hh signaling is the induction of target through the Gli cell line M2-10B4 cells (hereafter M2 cells) was stimulated with family of factors. Of the three family members, Gli2 purmorphamine (PM), a agonist of Hh signaling and Gli3 are the primary effectors for Hh-induced transcription (5). The M2 cells were shown to undergo osteoblastogenesis in and, thus, critical for embryogenesis (6–9). Gli1, however, itself response to Hh (30). Through a series of dosage and time-course a target of Gli2 and Gli3, is dispensable in the mouse embryo experiments, we found that 1 μM PM maximally induced the (7, 10). Biochemically, Gli2 is predominantly present in the full- length form that becomes a transcriptional in response Significance to Hh, whereas Gli3 exists preferentially as a C-terminally trun- cated transcriptional whose formation is inhibited by Hh Hedgehog (Hh) signaling is essential for embryonic bone for- (11–13). Overall, activation of Gli2 together with derepression of mation in mammals. However, how Hh signaling executes the Gli3 constitutes the primary mechanism for Hh-induced osteogenic function is not clear. We describe a positive feed- activation, whereas Gli1 plays a secondary role in amplifying the back mechanism whereby Hh-Gli2 signaling induces expression transcriptional response. of insulin-like growth factor 2 (Igf2), which signals via protein Indian hedgehog (Ihh) is indispensable for osteoblast de- kinases mTORC2 and Akt to stabilize Gli2 and potentiate Hh velopment in the endochondral skeleton of the mouse embryo induction of osteoblast differentiation. The Hh-Igf positive (14). Genetic studies of Smo have demonstrated that Ihh input is feedback loop may have general implications for Hh signaling directly required for osteoblast differentiation from the peri- in development and disease. chondrial progenitors in the long bone (15). Furthermore, forced Author contributions: F.L. designed research; Y.S. and J.C. performed research; Y.S., C.M.K., activation of Gli2 together with Gli3 deletion is sufficient to and F.L. analyzed data; and Y.S. and F.L. wrote the paper. replace Ihh in inducing osteoblast formation in the mouse em- The authors declare no conflict of interest. bryo, indicating that Gli2 activation and Gli3 derepression are This article is a PNAS Direct Submission. primarily responsible for Ihh-induced osteoblastogenesis (16). 1Present address: Department of Orthopaedic Surgery, Duke University School of Medi- The osteogenic effect of Gli2 and Gli3 is likely amplified by Gli1, cine, Durham, NC 27710. which has been shown to induce expression of osteoblast marker 2To whom correspondence should be addressed. Email: [email protected]. genes Alpl and Ibsp (17). However, how Ihh-Gli signaling acti- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vates the entire osteogenic program remains unresolved. 1073/pnas.1502301112/-/DCSupplemental.

4678–4683 | PNAS | April 14, 2015 | vol. 112 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1502301112 Downloaded by guest on September 26, 2021 mRNA levels of Gli1 and Ptch1, both Hh transcriptional targets, at 48 or 72 h of treatment (SI Appendix, Fig. S1). Importantly, 1 μM PM activated expression of several common osteoblast markers including Sp7, Alpl, and Bglap at those time points (SI Appendix, Fig. S1). Staining for Alpl activity or matrix mineral- ization confirmed the induction of osteoblast differentiation by 1 μM PM (Fig. 1A). Knockdown of Gli2, the primary effector for Hh-mediated transcriptional activation, not only reduced Gli1 induction, but also notably diminished the expression of the os- teoblast markers in response to PM (Fig. 1B). Thus, PM activates Hh-Gli signaling to induce osteoblast differentiation in M2 cells. To discover effector molecules downstream of Hh-Gli signal- ing, we performed RNA-seq (whole transcriptome shotgun se- quencing) experiments to compare the transcriptome of M2 cells with or without PM stimulation for 24 or 72 h. By examining all genes expressed at >1 read per kilobase of transcript per million reads mapped in at least one condition, and affected by PM by >twofold, we identified Igf2 and Igfbp5 being induced at 24 h, and Igf1, Igf2, Igfbp6, and Igfbp7 increased at 72 h. RT-qPCR (reverse transcription-quantitative polymerase chain reaction) experiments confirmed that PM significantly increased Igf2 mRNA by 24 h and reached the maximal induction of >20 fold by 48 h. The induction of Igf1 mRNA was less pronounced, reaching only ∼twofold after 72 h (Fig. 2A). Igfbp5 showed a twofold induction by 6 h, but Igfbp6 and Igfbp7 were also notably increased by 48 h (>twofold) (SI Appendix, Fig. S2). Furthermore, knockdown of Gli2 abolished Igf2 induction by PM at 24 h (Fig. 2B). Thus, Hh signaling up-regulates the expression of Igf signaling components. To test whether Hh activates Igf signaling, we monitored the phosphorylation of insulin receptor substrate 1 (Irs1) at S632/635 (equivalent of S636/639 in IRS1), which is stimulated by Fig. 2. Hh induces Igf-mTORC2 signaling through Gli2. (A) qPCR analyses in M2 cells treated with 1 μM PM or DMSO (Veh) for 6–72 h. (B) Effect of Gli2 insulin or Igf signaling (31). In keeping with the maximal in- knockdown on Igf2 mRNA level in response to PM for 24 h as assayed by duction of Igf2 at 48 h, PM notably increased both total- and qPCR. Ribosomal 18S rRNA used as internal control for all qPCR. *P < 0.05, phospho-Irs1 at 48 and 72 h (Fig. 2C). Recombinant human N-Shh n = 3. (C) Western blot for phospho-Irs1(S636/639) (P-Irs1) after 1 μMPM had a similar effect on the phosphorylation (SI Appendix,Fig.S3). treatment for 48 or 72 h. P-Irs1 is normalized to α-tubulin and fold changes Importantly, knockdown of Igf2, Igf1r, or Gli2 severely blunted were calculated from three independent experiments with SD indicated. the induction of P-Irs1 at 72 h by PM (Fig. 2 D and E). Taken (D and E) Effects of shRNA knockdown of Igf1r, Igf2 (D), or Gli2 (E) on P-Irs1 in response to PM treatment for 72 h. P-Irs1 is normalized to total Irs1, and fold changes were calculated from three independent experiments with SD indicated. (F and G) Effects of shRNA knockdown of Gli2 (F), Igf1r, or Igf2 (G) on P-Akt (S473) in response to PM treatment for 72 h. P-Akt (S473) is normalized to total Akt, and fold changes were calculated from three in- dependent experiments with SD indicated. Sh-Lacz was used as control.

together, the data indicate that Hh activates Igf signaling through up-regulation of Igf signaling components. We next investigated the intracellular pathway downstream of Igf1r in response to Hh signaling. In time-course experiments, we found that PM consistently increased phosphorylation of Akt at S473 [P-Akt (S473)] by 48 h, indicating activation of mTORC2 (SI Appendix, Fig. S4). However, PM did not induce mTORC1 activity, because phosphorylation of S6 or Eif4ebp1 was not different from the control at any of the time points examined (SI Appendix, Fig. S4A). Likewise, N-Shh activated mTORC2 but not mTORC1 after 48 or 72 h of treatment (SI Appendix, Fig. S4B). Importantly, knockdown of Gli2, Igf1r, or Igf2 greatly suppressed the induction of mTORC2 activity by PM, as mea- sured by P-Akt (S473) levels (Fig. 2 F and G). Thus, Hh signaling activates Igf-mTORC2 signaling. Fig. 1. Hh signaling induces osteoblast differentiation through Gli2 in M2 We then investigated whether Igf-mTORC2 signaling con- cells. (A) Alkaline phosphatase activity (ALP) in M2 cells treated with 1 μM tributes to Hh-induced osteoblast differentiation in vitro. Knock- PM or DMSO (Veh) for 72 h. Alizarin Red staining was performed after ad- down of Igf2 abolished osteoblast differentiation in either M2 cells ditional 8 d of incubation in mineralization medium. (B) Effects of shRNA or primary bone marrow stromal cells (BMSCs) in response to knockdown of Gli2 (sh-Gli2) on mRNA levels of indicated genes as assayed by

qPCR. Cells were infected with lentivirus expressing sh-Gli2 or sh-Lacz for 24 h PM, as indicated by the lack of Sp7, Alpl, and Bglap induction BIOLOGY μ (Fig. 3A and SI Appendix,Fig.S5). Knockdown of Igf1r also before further incubation with or without 1 M PM for 72 h. Ribosomal 18S DEVELOPMENTAL rRNA was used as internal control for qPCR. *P < 0.05, n = 3. suppressed PM-induced osteoblast differentiation, but knockdown

Shi et al. PNAS | April 14, 2015 | vol. 112 | no. 15 | 4679 Downloaded by guest on September 26, 2021 expressing Flag-tagged full-length Gli2 (Flag-Gli2) in response to doxycycline (Dox), and then treated the cells with Igf2. Igf2 had no effect on Gli2 mRNA levels (including both endogenous Gli2 and exogenous Flag-Gli2), but notably increased the protein level of Flag-Gli2, indicating that Igf2 may suppress Gli2 turn- over (SI Appendix, Fig. S9 A and B). In contrast, Igf2 did not affect the protein level of Flag-Gli3 in a similar experiment (SI Appendix, Fig. S9C). To determine the potential effect of Igf2 on the half-life of Gli2, we blocked de novo protein synthesis with cyclohexamide after the induction of Flag-Gli2 expression with Dox, and monitored the abundance of Flag-Gli2 as a function of time with or without Igf2 treatment. These experiments revealed that Igf2 prolonged the half-life of Flag-Gli2 from <6hto>24 h, and that the stabilizing effect of Igf2 was completely abolished by the Akt inhibitor MK2206 (Fig. 4D). Examination of the mouse Gli2 protein sequence revealed a consensus phosphorylation site for Akt, R225KRALS230 (32). Alanine substitution of S230 abolished the stabilizing effect of Igf2 on Gli2 (Fig. 4E). Finally, to confirm the effect of Igf2 on endogenous Gli2, we generated an against a peptide within the C-terminal half of the mouse Gli2 (SI Appendix, Fig. S10) and found that Igf2 stabilized the endogenous full-length Gli2 protein (Fig. 4F). Thus, Igf2 markedly suppresses Gli2 turnover likely through direct phos- phorylation by Akt.

Deletion of Igf1r in Hh-Responsive Cells Impairs Bone Formation in Vivo. To investigate the contribution of Igf signaling to Hh-induced osteoblast differentiation in vivo, we sought to disrupt Igf signaling Fig. 3. Igf2-mTORC2-Akt signaling is required for Hh-induced osteoblasto- in the Hh-responsive osteogenic cells in the mouse embryo by genesis. (A) Effects of Igf2 knockdown on mRNA levels of indicated genes in using the Gli-CreERT2 mouse line. To establish the effective- response to PM for 72 h, as assayed by qPCR. (B and C) Effect of Rictor T2 knockdown (B) or MK2206 (C) on P-Akt (S473) assayed by Western blot, and ness of strategy, we first crossed Gli-CreER with the mT/mG on mRNA levels of osteoblast markers assayed by qPCR. (D) Effects of reporter mouse and exposed the embryos to tamoxifen (TM) from MK2206 on ALP or Alizarin Red staining in response to PM. Ribosomal 18S embryonic day (E)13.5 until E16.5 when they were harvested. In T2 rRNA used as internal control for all qPCR. *P < 0.05, n = 3. the long bones of the Gli-CreER ; mT/mG embryo, numerous green cells were observed within the osteogenic perichondrium and the cartilage, indicating that Gli-CreERT2 effectively targets the of insulin receptor (Insr) had no effect (SI Appendix,Fig.S6). Furthermore, knockdown of the mTORC2-specific component Rictor greatly diminished PM-induced osteoblast differentiation (Fig. 3B). Pharmacological inhibition of all mTOR activity with Torin1 exerted even greater inhibition on differentiation (SI Ap- pendix,Fig.S7). Finally, inhibition of the mTORC2 target Akt with MK2206 abolished PM-induced mRNA expression of Sp7, Alpl, and Bglap, as well as Alpl staining and matrix mineralization (Fig. 3 C and D). Thus, Hh signaling activates the Igf2-mTORC2- Akt axis to induce osteoblast differentiation.

Igf2 Synergizes with Hh Signaling via Akt-Dependent Gli2 Stabilization. We next pursued how Igf2-mTORC2-Akt signaling mediates Hh-induced osteoblastogenesis. Igf2 by itself did not induce the expression of Sp7, Alpl, or Bglap in M2 cells, but synergistically increased the mRNA levels of these markers when used in com- bination of suboptimal levels (e.g., 0.5 μM) of PM (Fig. 4A). In- terestingly, in such settings, Igf2 also boosted the expression levels of Hh target genes Gli1 and Ptch1 compared with PM alone, al- though Igf2 itself did not induce these genes (Fig. 4A). Conversely, knockdown of Igf2 or Igf1r reduced the mRNA levels of Gli1 and Ptch1 in response to PM (Fig. 4B and SI Appendix,Fig.S8A). Likewise, inhibition of mTORC2 with either Torin1 or by knocking down Rictor severely diminished the induction of Gli1 by PM (SI Appendix,Fig.S8B and C). Finally, inhibition of Akt with MK2206 also blunted the Hh transcriptional response (Fig. 4C). Thus, Igf2- Fig. 4. Igf2 synergizes with Hh signaling via Akt-dependent Gli2 stabiliza- μ mTORC2-Akt signaling stimulates osteoblast differentiation by tion. (A) Synergistic induction of genes by 0.5 M PM or 100 ng/mL Igf2, as augmenting the transcriptional response to Hh signaling. assayed by qPCR after 72 h of treatment. (B and C) Effects of Igf2 knock- down (B) or MK2206 (C) assayed by qPCR after 72 h of PM treatment. *P < To determine how Igf2-mTORC2-Akt enhances transcrip- 0.05, n = 3. (D) Effects of Igf2 and MK2206 on Flag-Gli2 turnover, as mea- tional activation by Hh, we examined the potential effect of Igf sured by Western blot. *P < 0.05, n = 3. (E) Effect of Igf2 on Flag-Gli2-S230A signaling on Gli2, the primary effector for Hh-induced gene turnover. (F) Effect of Igf2 on endogenous Gli2 stability. All quantification is activation. To this end, we infected M2 cells with lentiviruses from three independent experiments. CHX, cyclohexamide.

4680 | www.pnas.org/cgi/doi/10.1073/pnas.1502301112 Shi et al. Downloaded by guest on September 26, 2021 cells known to respond to Ihh during long bone development (Fig. wild-type littermates (Fig. 5 F and G). In contrast, the level of full- 5A). We next generated Gli-CreERT2;Igf1rf/f embryos (CKO) and length Gli3 was largely normal in the CKO samples (Fig. 5G). In induced Igf1r deletion with the same TM regimen. Immunofluo- summary, Igf signaling is necessary to potentiate osteoblastogenesis rescence staining confirmed that Igf1r was notably reduced in both in Ihh-responsive cells during long bone development. perichondrial cells and chondrocytes in the long bones of E16.5 embryos (SI Appendix,Fig.S11). Importantly, whole-mount skel- Discussion etal staining revealed that the bone collars of all long bones in the We have uncovered a positive feedback mechanism between Hh CKO embryo were notably shorter than their normal counterparts, and Igf signaling during osteoblast differentiation. In particular, although the total length of each element appeared to be similar Hh signaling induces transcription of multiple members of the across genotypes (Fig. 5B). Quantification indicated that the - Igf system, most notably Igf2, resulting in activation of the tive length of bone collar versus the entire element was reduced by mTORC2-Akt signaling axis. Akt, in turn, stabilizes full-length 10–20% in the CKO embryo, indicating that ossification was sup- Gli2, likely through phosphorylation of S230, thereby amplifying pressed (Fig. 5C). The bone collar deficit persisted at E18.5 (SI the transcriptional output of Hh signaling. Supporting the Appendix,Fig.S12). Staining of the long bone sections with the von physiologic relevance of the Hh-Igf positive feedback loop, cell- Kossa method identified a marked decrease in bone collar min- autonomous removal of Igf signaling in Ihh-responding cells diminishes osteoblast differentiation in the mouse embryo. This eralizationintheCKOembryo(Fig.5D). Furthermore, in situ finding not only sheds light on how Hh signaling induces osteo- hybridization on sections showed that the mRNA levels of osteo- blastogenesis, but also adds another dimension to the role of Igf blast markers Runx2, Sp7, Alpl, and the Hh target gene Ptch1 were signaling in bone formation. diminished in CKO, although Ihh expression was relatively normal The mechanism whereby Hh activates osteoblast differentia- (Fig. 5E). However, chondrocyte proliferation or hypertrophy was tion remains to be fully elucidated. Our data have demonstrated relatively normal in E14.5 CKO embryos after 3 d of TM induction an important role for Igf signaling in Hh-induced osteoblasto- (SI Appendix,Fig.S13). Western blot analyses confirmed that in genesis, but it is also clear that Igf signaling cannot replace Hh to the limbs of the E14.5 CKO embryos, the levels of Igf1r, Gli2, and initiate the osteogenic program. In contrast to Ihh deletion that P-Akt (S473) were notably reduced compared with those in the abolishes bone formation altogether in the endochondral skele- ton, removal of Igf1r impairs but does not prevent the process (33). Data from the present study support the model wherein Igf signaling amplifies Gli-mediated transcriptional induction to pro- mote osteoblast differentiation. This model predicts that certain osteogenic genes are induced not immediately by Hh but only after subsequent activation of Igf signaling, a process occurring between 24 and 48 h of PM treatment in M2 cells. Indeed, our RNA-seq experiments identified several transcription factors be- ing induced after 72 but not 24 h of PM treatment; these factors include Dlx5, Dlx6, Sp7, Msx1, and Ets2, all of which have been implicated in osteoblast differentiation (34–37). Future studies are necessary to determine the contributions of those factors to os- teoblast differentiation in response to Hh signaling. The mechanism whereby Hh signaling induces Igf2 expression is not clear at present. We show that Gli2 is necessary for Hh to induce Igf2 mRNA. This result, along with the previous report that Gli1 overexpression induces Igf2 in C3H10T1/2 cells, raises the possibility that Igf2 might be a direct target of Gli activators (38). However, unlike the known Gli target genes such as Gli1 that show activation by 6 h of Hh stimulation, Igf2 is not induced until after 24 h (SI Appendix, Fig. S1). Moreover, although a putative Gli binding site is identified in the 3′UTR of the Igf2 gene, immunoprecipitation experiments with cells over- expressing Flag-tagged Gli2 have failed to reveal Gli2 binding to the site. We have considered the possibility that other secreted factors such as BMP, Wnt, or PTHrP may mediate the induction of Igf2 by Hh, but the RNA-seq experiment did not detect any significant increase in their expression after 24 h of PM stimulation. Thus, the induction of Igf2 by Gli2 appears to be through an indirect and yet Fig. 5. Deletion of Igf1r in Hh-responsive cells impairs bone formation in vivo. (A) Detection of GFP by immunofluorescence on a tibial section from an unknown mechanism. E16.5 Gli1-CreERT2; mT/mG mouse embryo that received daily tamoxifen be- We have identified S230 of Gli2 as a critical mediator for Igf2 ginning at E13.5. H, hypertrophic chondrocytes; P, perichondrium; PH, pre- to potentiate Hh signaling. In our model, upon activation by Igf2, hypertrophic chondrocytes; S, primary spongiosa. (B and C) Skeletal staining Akt phosphorylates and stabilizes full-length Gli2 that requires of long bones and quantification of relative bone collar length in E16.5 wild- Hh signaling for activation. Previous work has identified clusters type (WT) versus Gli1-CreERT2; Igf1rf/f (CKO) littermate embryos. Vertical lines of adjacent serine residues whose sequential phosphorylation demarcate the ends of the bone collar; the horizontal lines denote the deficit by PKA and Gsk3 controls Gli2 turnover (39). That study also in the mutant (CKO). *P < 0.05, n = 5. F, femur; H, humerus; R, radius; T, tibia; reported that alanine substitutions of the PKA, but not Gsk3 U, ulna. (D) Von Kossa staining on longitudinal tibial sections in WT versus or Akt phosphorylation sites, abolished the inhibitory effect of CKO littermates at E16.5. Green boxed areas shown at a higher magnification in Insets.(E) In situ hybridization on tibial sections of E16.5 embryos. Signal is dominant negative Akt on Gli2-mediated transcription, although in red. (F and G) Western blot analyses of protein extracts from limbs of E14.5 it did not examine the stability of Gli2. It is possible that Akt-

WT versus CKO littermates with TM treatments beginning at E11.5. Each lane mediated S230 phosphorylation precludes PKA-mediated phos- BIOLOGY α

is from a separate embryo. Intensity normalization is against -tubulin, except phorylation, thus stabilizing Gli2. Moreover, the full effect of Akt DEVELOPMENTAL for P-Akt (S473) against total Akt. on Gli2 stability may involve both the direct regulation as reported

Shi et al. PNAS | April 14, 2015 | vol. 112 | no. 15 | 4681 Downloaded by guest on September 26, 2021 here and indirect control via phosphorylation and inactivation of Cell Culture. M2-10B4 cells (CRL-1972; ATCC) were maintained in RPMI (cat- Gsk3 (40). Future studies are necessary to determine whether and alog no. 11875-085, Gibco) with 10% FBS (Gibco) as per instructions. Unless × 4 2 how Akt-mediated phosphorylation of Gli2 affects its phosphor- otherwise indicated, M2 cells were seeded at the density of 1 10 cells per cm , 48 h before experiments. Primary cultures of BMSCs were harvested from ylation by PKA or Gsk3. the femur and the tibia of 6-wk-old mice and cultured in α-MEM containing It is worth noting that deletion of Igf1r in Hh-responsive cells 10% FBS (Gibco), with a change of medium at day 3. Cells were dissociated at did not grossly affect cartilage development. This observation is day 7 and reseeded at 1 × 105 cells per cm2 for qPCR, ALP staining or miner- consistent with our previous finding that Ihh and Igf control the alization assays at appropriate time points. To express Flag-tagged Gli2 proteins linear growth of the skeleton largely independently (29). In ad- in M2 cells, the mouse Gli2 full-length cDNA, either wild type or the S230A dition, previous studies have demonstrated that Ihh controls mutant form, was cloned into the Fuw-tetO vector (plasmid 20723; Addgene) cartilage development chiefly through derepression of Gli3, but and retrovirus was produced according to standard protocol. A retrovirus induces osteoblastogenesis via both Gli3 derepression and Gli2 producing Flag-Gli3 (full-length) was made in the same way. Then M2-10B4 cells were infected with FuW-rtTA (plasmid 20342; Addgene) virus, together with activation (16, 41, 42). Here, we show that Igf signaling stabilizes Fuw-tetO-FlagGli2 or Fuw-tetO-FlagGli3 virus. Gli2 expression was induced with full-length Gli2 but not Gli3. Thus, the relative contribution of addition of doxycycline (100 nM) to the media. The recombinant human Shh Gli2 versus Gli3 likely determines the functional relevance of the (464-SH-025) and Igf2 (792-MG-050) were purchased from R&D Systems. Pur- Hh-Igf feedback mechanism in specific physiological contexts. morphamine (540223; Millipore), MK2206 (S1078; Selleckchem), Torin1 Beyond osteoblastogenesis, Hh and Igf may functionally in- (4247; Tocris Biosciences) were purchased. Gli2 shRNA plasmid (Mission + − teract in other processes. Most notably, in the Ptch1 / mouse shRNA SHCLND-NM_001081125) was purchased from Sigma. Lentiviral shRNA model for and rhabdomyosarcoma, Igf2 was up- targeting vectors were purchased from Washington University shRNA con- regulated and necessary for tumor formation (43). Similarly, sortium. Targeted sequences are listed in SI Appendix,TableS1. cooverexpression of Igf2 or activated Akt with Shh synergistically Osteoblast Differentiation Assays. Alkaline phosphatase staining was as de- induces medulloblastoma in mice (44). Mechanistically, Igf-PI3K scribed (53). When inhibitors were used, cells were pretreated with the in- and Hh signaling appeared to converge on Nmyc1 to promote hibitor in normal growth medium for 1 h. When shRNA lentiviruses were proliferation of cerebellar neuronal precursors (45). Moreover, Igf used, cells were infected with respective shRNA lentivirus for 24 h before synergized with Shh in promoting somite in explant subsequent treatments. For mineralization assays, confluent cells were incubated cultures (46). Overall, Hh and Igf signaling exhibit synergistic in the presence of 50 mg/mL ascorbic acid and 50 mM β-glycerophosphate for 8 d interactions both in normal development and during pathogenesis. and then stained with Alizarin Red S. Materials and Methods Western Blot Analyses. Protein extracts from cells were prepared in radio- immunoprecipitation assay buffer containing phosphatase and proteinase High-Throughput RNA Sequencing. M2 cells were treated with or without 1 μM inhibitors (Roche). Total protein content was determined by the bicinchoninic PM for 24 or 72 h before total RNA was isolated by using the RNeasy kit with acid method (Pierce). Membranes were imaged with Molecular Imager on-column DNase treatment (Qiagen). Poly-A RNA was isolated from 10 μgof ChemiDoc XRS+ System (Bio-Rad). Quantification of Western blots was total RNA by using Oligo-dT beads, followed by fragmentation and reverse performed with ImageLab software. The monoclonal against transcription. The double-stranded cDNA was used to generate a library with phospho-IRS1(S636/S639) (2388), total-IRS1 (3407), phospho-AKT(S473) sequencing adapters added to the ends of DNA fragments, and the resulting (9271), total-AKT (9272), phospho-S6 (2215), total-S6 (2217), phospho- library was sequenced by using the Illumina HiSeq-2000 as single reads extending Eif4ebp1 (T37/46) (2855), total-Eif4ebp1 (9644), Rictor (2140) and β- 42 bases ( Technology Access Center, Washington University School of (4970) were purchased from Technology. The antibody against Medicine). The raw data were demultiplexed and aligned to the reference ge- Gli3 (ab6050) was from Abcam, and the M2 antibody (catalog no. F1804) nome (mm9) by using TopHat. Partek Genomic Suite was used to assemble against Flag was from Sigma. The monoclonal antibody against α-tubulin (sc- transcripts and analyze expression. 8035) and the polyclonal antibody against Igf1r (sc-713) were obtained from Santa Cruz Biotechnology. HRP-conjugated anti-rabbit (NA934V) or anti- f/f T2 Mouse Studies. Igf1r , mT/mG, and Gli1-CreER mice have been reported mouse secondary antibody (sc-2005) was purchased from GE Healthcare or (26, 47, 48). The Studies Committee at Washington University in Santa Cruz Biotechnology, respectively. The Alexa Fluor 488 donkey anti- St. Louis reviewed and approved all mouse procedures used in this study. The rabbit IgG (H+L) antibody was obtained from Invitrogen. Gli1-CreERT2;Igf1rf/f or Gli1-CreERT2;mT/mG pregnant female mice were ad- ministered tamoxifen dissolved in corn oil (4 mg/30 g of body weight) by oral Quantitative PCR. RNA was isolated from whole cells with QIAGEN RNeasy kit gavage daily for durations as indicated. Whole-mount skeletal staining was (74104) and was transcribed into cDNA by using iScript cDNA synthesis kit performed with a protocol based on McLeod (49). To assess skeletal pheno- (Bio-Rad). Fast-start SYBR Green (Bio-Rad) and 0.1 mM primers were used in types on sections, embryonic limbs were harvested in PBS and fixed in each reaction. Ribosomal 18S RNA was used for normalization. PCR was 10% (vol/vol) buffered formalin overnight at room temperature. The fixed performed with Step-One machine (ABI). The primer sequences are provided limbs were then processed and embeddedinparaffinbeforesectioningat6μm. in SI Appendix, Table S2. Limbs from E18.5 embryos were decalcified in 14% (wt/vol) EDTA/PBS (pH 7.4) for 48 h after fixation and before processing. In situ hybridization was per- Production of Gli2 Antibody. A monoclonal antibody specific for full-length 35 formed by using S-labeled riboprobes as described (15, 42, 50, 51). The Gli2 was generated against a peptide within the C-terminal half of mouse Gli2 chondrocyte proliferation rate in embryos was assessed by immunodetection according to proprietary techniques (Abmart). The purified antibody was of BrdU incorporation on sections of the embryos (Invitrogen) (52). The validated by Western blot analyses for detection of overexpressed Gli2, and percentage of labeled cells was calculated from medial sections of three pairs for loss of endogenous Gli2 following shRNA knockdown. of wild-type versus mutant littermates. Igf1r immunofluorescence staining was performed on paraffin sections after antigen retrieval with citrate buffer Statistical Analyses. All quantitative data are presented as mean ± SD with and blocking with 5% (vol/vol) normal goat serum in Tris buffered saline with a minimum of three independent samples. Statistical significance is de- Tween. Sections were incubated with Igf1r antibody (sc-713; Santa Cruz termined by Student’s t test. Biotechnology) at 1:100 dilution overnight at 4 °C, and then Alexa Fluor 488 donkey anti-rabbit IgG (H+L) antibody (A11371; Invitrogen) at 1:300 dilution ACKNOWLEDGMENTS. This work is supported by NIH Grant R01 DK065789 at room temperature for 1 h. (to F.L.).

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