Thyroid-stimulating hormone induces a Wnt-dependent, feed ... · Thyroid-stimulating hormone induces a Wnt-dependent, feed-forward loop for osteoblastogenesis in embryonic stem cell

Thyroid-stimulating hormone induces a Wnt-dependent,feed-forward loop for osteoblastogenesis in embryonicstem cell culturesRamkumarie Balirama, Rauf Latifa, Joshua Berkowitza, Simon Frida, Graziana Colaiannib, Li Sunb, Mone Zaidib,and Terry F. Daviesa,b,1

aThyroid Research Laboratory and bMount Sinai Bone Program, Mount Sinai School of Medicine, and James J. Peters VA Medical Center, New York, NY 10029

Edited* by Maria I. New, Mount Sinai School of Medicine, New York, NY, and approved August 16, 2011 (received for review June 24, 2011)

We have shown that the anterior pituitary hormone, thyroid-stimulating hormone (TSH), can bypass the thyroid to exert a directprotective effect on the skeleton. Thus, we have suggested thata low TSH level may contribute to the bone loss of hyperthyroidismthat has been attributed traditionally to high thyroid hormonelevels. Earlier mouse genetic, cell-based, and clinical studiestogether have established that TSH inhibits osteoclastic boneresorption. However, the direct influence of TSH on the osteoblasthas remained unclear. Here, we have used a model system de-veloped frommurine ES cells, induced to formmature mineralizingosteoblasts, and show that TSH stimulates osteoblast differentia-tion primarily through the activation of protein kinase Cδ and theup-regulation of the noncanonical Wnt components frizzled andWnt5a. We predict that a TSH-induced, fast-forward short loop inbone marrow permits Wnt5a production, which, in additionto enhancing osteoblast differentiation, also stimulates osteopro-tegerin secretion to attenuate bone resorption by neighboringosteoclasts. We surmise that this loop should uncouple bone for-mation from bone resorption with a net increase in bone mass,which is what has been observed upon injecting TSH.

Until recently, thyroid-stimulating hormone (TSH) wasthought solely to regulate thyroid follicle development and

thyroid hormone secretion (1). Although radio-ligand bindingand mRNA studies had suggested that TSH receptors (TSHRs)were expressed more ubiquitously (2, 3), it was only in 2003 thatwe definitively established that TSHRs were localized to bonecells and that absent TSHR signaling caused high-turnover boneloss (4). Subsequent rodent and clinical studies have sincepointed to a role for TSHRs in normal physiology, as well as inthe pathophysiology of bone loss in the hyperthyroid state (5–9).A recent study, in which a woman with isolated TSH deficiencydeveloped not only myxedema coma, but also severe osteopo-rosis, is more evidence for a fundamental role for TSH in skeletalhomeostasis (10). Less compelling, but nonetheless important,is that individuals with activating TSHR polymorphisms but withnormal thyroid function have a higher bone mass than matchedcontrols (11).TSH inhibits bone resorption directly by acting on osteoclastic

TSHRs (4, 12), as well as indirectly by suppressing the pro-duction of the osteoclastogenic cytokine tumor necrosis factor-α(TNF-α) from macrophages (13). However, when administeredin vivo, TSH not only prevents bone loss via an antiresorptiveaction (14), but also stimulates bone formation in certain rodentmodels, such as aged rats, to restore the lost bone even 28 wkpost ovariectomy (14, 15). These studies have led to the specu-lation that, in addition to its osteoclast-inhibitory actions seen inmice, rats, and humans, TSH may also stimulate bone formationthrough a direct action on the osteoblast, which we have shownpossesses TSHRs in abundance (4).We know that children with congenital hypothyroidism have

runted skeletons. Although this skeletal defect has been attrib-uted solely to low thyroid hormone levels, the emerging function

of TSH in skeletal physiology begs the question whether TSH alsoplays a role in skeletal morphogenesis and bone growth. In otherwords, can a high TSH cause premature ossification in growingchildren, or at least contribute to it? Hence, our use of an ES cellmodel, which we have found expresses TSHRs (16). That a gly-coprotein hormone receptor, such as the TSHR, should appear soearly in development is a testament to its general importancein embryology.Hence, the studies described here serve two distinct, but related

purposes. First, we describe an experimental system in whichTSHR-positive ES cells can be induced to form mature, miner-alizing osteoblasts. Second, and of equal importance, is that weshow clearly that TSH can functionally stimulate the osteoblastdifferentiation in these ES cell cultures. These data suggest thatTSH could indeed be a hormonal (or local) regulator of boneaccretion during skeletal morphogenesis and growth.

ResultsWe first studied the effect of incubating murine ES cells with os-teogenic differentiation factors on the formation of osteoblastcolonies during a 30-d culture.We found a significant decline in theexpression of the stem cell renewal genes Rex-1, Oct-4, and Sox-2(Fig. 1A); this suggested that ES cells had begun to lose their“stemness.” In parallel, the cultures began to display alkalinephosphatase (ALP)-positive colonies, colony-forming units-fibro-blastoids (CFU-fs) (Fig. 1B). PCR revealed the expression of bothearly and late osteoblast differentiation genes, namely ALP, type 1collagen, and osteocalcin (Fig. 1C). In separate experiments, ma-ture mineralized colonies, colony forming units-osteoblasts (CFU-obs), were seen with von Kossa (Fig. 3A) and Alizarin red staining(Fig. 3B). Mineralization was confirmed by increased calcium andphosphorous content of the colonies, respectively (Fig. 3C andD).We next studied whether the TSHR was expressed in these

cultures, and if so, whether its activation affected osteoblast dif-ferentiation. Fig. 2A shows TSHR mRNA in ES cells throughoutthe 30-d cultures. The addition of TSH appeared to dampenTSHR expression. TSHR protein expression was confirmed byflow cytometry using the anti-TSHR monoclonal antibody M1directed to residues 381–385 (Fig. 2B). Seventy-three percent ofcells displayed TSHR expression.

Author contributions: R.B. and T.F.D. designed research; R.B., R.L., J.B., S.F., and L.S.performed research; S.F., G.C., and T.F.D. contributed new reagents/analytic tools; R.B.,R.L., L.S., M.Z., and T.F.D. analyzed data; and R.B., R.L., G.C., L.S., M.Z., and T.F.D. wrotethe paper.

Conflict of interest statement: MZ is a named inventor of a pending patent applicationrelated to the use of TSH in the inhibition of TNF activity. This patent has been filed by theMount Sinai School of Medicine (MSSM). In the event the patent is licensed, MZ would beentitled to a share of any proceeds MSSM receives from the licensee.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110286108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1110286108 PNAS | September 27, 2011 | vol. 108 | no. 39 | 16277–16282

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

Nov

embe

r 6,

202

0

Activation of the TSHR in ES cells enhanced osteoblast dif-ferentiation markedly. Von Kossa- and Alizarin red-positiveCFU-obs increased significantly in the presence of TSH comparedwith control cultures containing differentiation medium only (Fig.3 A and B). Enhanced mineralization was confirmed by calciumand phosphate measurements (Fig. 3 C and D). Importantly,however, TSH triggered a dramatic, up to 200-fold, concentra-tion-dependent increase in type 1 collagen. It also enhanced theexpression of ALP and osteocalcin (Fig. 3E). The data togetherdemonstrate that TSH stimulates osteoblastogenesis by acting onthe TSHR in differentiating ES cell cultures. This pro-osteoblasticaction of TSH is consistent with studies from our group andothers, wherein TSH injections are anabolic in vivo. Notably,injected TSH restores the lost bone post ovariectomy andenhances bone formation in calcein-labeling studies (14, 15).To probe the mechanism of TSH-induced osteoblastogenesis,

we studied the expression of molecules involved in Wnt signal-ing, a dominant pathway regulating bone formation and bonemass. Fig. 4A shows a small enhancement in the expression ofthe canonical Wnt receptor LRP5 upon differentiation, but thiswas attenuated by TSH, confirming previous results (4). LRP6

remained relatively unaffected by differentiation or by TSH (Fig.4B). The target canonical Wnt transducer β-catenin likewiseremained relatively unaltered upon differentiation and by TSH(Fig. 4C). In contrast, the noncanonical coreceptor frizzled (Frz)was up-regulated dramatically by TSH (Fig. 4D), in a concentra-tion-dependent manner (data not shown). Likewise, quantitativePCR and Western blotting showed a strong induction by TSH ofthe noncanonical agonist Wnt5A (Fig. 4 E and F). The datademonstrated that the noncanonical arm of the Wnt signalingpathway, consisting of Frz andWnt5a, was the primary transducerof TSH effects on osteoblastogenesis. Consistent with this path-way, the production of osteoprotegerin (OPG) was also enhancedas a function of osteoblast differentiation and with TSH by ∼50-fold (Fig. 4F). This increase was confirmed on Western blotting(Fig. 4G).Finally, we sought to explore proximal pathways: notably,

whether the induction of osteoblastogenesis by TSH was medi-ated by PKA or PKCδ, both of which are known to be downstreamof TSHR activation, but only one of which, namely PKCδ, hasbeen implicated in mediating osteoblastogenesis (17). TheWestern blot in Fig. 5A shows that PKCδ phosphorylation wasmarkedly enhanced upon ES cell differentiation and with TSH,whereas PKA phosphorylation was not. To assess a possiblefunction for PKCδ in TSH action, we studied whether the knownPKCδ inhibitor rotterlin could inhibit TSH-induced osteoblasto-genesis. Thus, whereas TSH stimulated expression of the differ-entiation genes osteopontin and type 1 collagen, rotterlininhibited this response (Fig. 5 B and C). This established thatPKCδ was a dominant downstream mediator of TSH-inducedosteoblastogenesis.

DiscussionThese results underscore important concepts relating to osteo-blast differentiation from pluripotent ES cells. First, we find thatmature, mineralizing osteoblasts can indeed form from ES cellswithout the intermediacy of embyroid bodies, which is consistentwith previous reports (16). This pattern is also consistent with thereduced expression of stem cell renewal genes and with the en-hanced expression of osteoblast marker genes. Second, the dataestablish that TSH stimulates osteoblastic differentiation fromES cells. This TSH-induced osteoblastogenesis is concordantwith, and might in fact explain, the anabolic action of TSHin vivo. Third, we show that TSH potently stimulates the pro-duction of OPG from ES cell-derived osteoblasts. This obser-vation is supported by data from primary cultures, whereinmature osteoblasts synthesize OPG and immature cells producemainly RANKL (18).

Fig. 1. Murine ES cell cultures can be induced to form osteoblasts. ES cell culture in differentiation medium (DF) (Materials and Methods) resulted in (A)a marked reduction in the expression of the stem cell marker genes Rex-1, Sox-2, and Oct-4 at day 30 compared with day 2 (by qPCR). *P < 0.005, **P < 0.0001,***P < 0.0004. (B) The appearance of alkaline phosphatase (ALP)-positive colonies and CFU-fs. (C) The expression of osteoblast genes, namely ALP, type 1collagen, osteocalcin (OC), and osteoprotegerin (OPG). Data are representative of two separate experiments.

Fig. 2. TSH receptors (TSHRs) are expressed in differentiating murine ES cellcultures. RT-PCR (A) and flow cytometry (B) were performed on ES cell cul-tures after 30 d of incubation in differentiation medium (DF) (Materials andMethods). (A) DF stimulated, with or without TSH (10 mU/mL) and TSHRmRNA expression in long-term cultures. FRTL5 cells treated with 5 hormonewas used as the positive control and GAPDH as the loading control. (B)Approximately 73% of cells were TSHR-positive when stained with anti-TSHRantibody (M1) (blue) compared with an isotype IgG control (red) (Materialsand Methods).

16278 | www.pnas.org/cgi/doi/10.1073/pnas.1110286108 Baliram et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 6,

202

0

At the mechanistic level, we show that the action of TSH isexerted primarily through the noncanonical Wnt pathway down-stream of PKCδ. Wnt signaling is known to increase bone massusing two separate mechanisms: directly, by enhancing osteo-blastogenesis and bone formation and, indirectly, by stimulatingthe production of OPG from osteoblasts, which, in turn, inhibitsRANK-L-induced osteoclastogenesis and bone resorption (19).Of note is that ES cell-derived osteoblasts produce abundantOPG that is further enhanced by TSH. This suggests that OPGmay, at least in part, be responsible for the indirect action of TSHin inhibiting bone resorption and increasing bone mass. That said,TSH also inhibits osteoclasts directly and, in addition, suppressesthe production of TNFα, a proresorptive cytokine (4, 13).The 19 secreted Wnts bind to 1 of 10 Frz receptors and one

of two LRPs (20). Of the four known intracellular signalingpathways, the best characterized is the canonical Wnt/β-cateninpathway that signals through LRP5/6, GSK-3β, and β-catenin toultimately activate the nuclear transcription complex lymphoidenhancement factor/T cell factor (20). Gain-of-function muta-tions of this pathway increase osteoblastogenesis, bone formation,and bone mass (21). Loss-of-function mutations cause diseasescharacterized by bone loss, such as the osteoporosis-pseudo-glioma syndrome (22). Our results show modest increases inLRP5, and no increases in LRP6 or β-catenin expression. LRP5levels dropped in response to TSH, which might suggest a modestinhibitory effect of TSH on canonical signaling. This is not un-expected, however, as (i) TSH decreased osteoblastogenesis inprimary osteoblast cultures from adult mice, and (ii) LRP5 ex-pression was elevated in osteoblasts from TSHR−/− mice (4).In contrast to the canonical pathway, mutations of the mo-

lecular components of the noncanonical pathway, abundantlyexpressed in bone cells, impair bone formation during skele-tal development (23). In differentiating ES cells, the alreadyhigh Frz levels were further up-regulated with TSH to ∼50-foldof basal. Furthermore, the prototypical agonist for noncan-

onical Wnt-signaling Wnt5a was dramatically enhanced byboth differentiation and TSH. Wnt5a is known to increase bonemass and to drive OPG production, mainly from mature osteo-blasts (24).Finally, TSH activated PKCδ, rather than PKA, in ES cell cul-

tures. Importantly, rotterlin, a specific PKCδ inhibitor, stronglyattenuated the osteoblastogenic response to TSH. This suggestedthat PKCδ mediated, at least in part, the actions of TSH onthe osteoblast. Of note is that PKCδ is downstream of parathy-roid hormone effects on osteoblast differentiation (25), and also, -together with Runx2, mediates anabolic responses to connexin-43and FGF-2 (26). Thus, the stimulation by TSH of PKCδ phos-phorylation further confirms the induction of an anabolic pathway.In summary, we show that ES cells are fully capable of dif-

ferentiating into mature mineralizing osteoblasts; that TSHpromotes osteoblast differentiation; that TSH stimulates OPGproduction, which may indirectly affect osteoclastogenesis; thata noncanonical pathway, mainly involving Frz and Wnt5a, me-diates the osteoblastogenic effect of TSH; and that PKCδ isa downstream mediator of TSH action. We thus propose a shortfeed-forward loop in which TSH stimulates Wnt5a productionthat not only enhances osteoblastogenesis, but also producesOPG, which, in turn, dampens osteoclastic resorption to furtherincrease bone mass (Fig. 6). Because osteoblasts and osteoclastsare in close anatomical proximity, and because their functionsare tightly coupled in time and space (18), it is possible that TSHuncouples bone formation from bone resorption, using Wnt5a.The question remains, however, whether systemic, pituitary-de-rived, TSH is responsible for this modulation or whether local,bone marrow TSH, such as a TSH-β subunit variant (27), is thekey to this permissive function.

Materials and MethodsCell Culture. Mouse ES cell line (WT 9.5) was maintained in a permanent plu-ripotent state with the addition of leukemia inhibitory factor (LIF) (10 ng/mL;

P<0.01

P<0.05

P<0.001

P<0.01

P<0.05

P<0.05

P<0.05

A

B

C D

E

Fig. 3. TSH stimulates osteoblastogenesis and mineralization. (A and B) Von Kossa-positive and Alizarin-positive colony formation in ES cell cultures in-cubated in differentiating medium (DF) for 30 d (Materials and Methods). (a) Untreated and (b) enhanced mineralization with differentiation and (c) in-fluence of TSH (1 mU/mL). TSH failed to induce differentiation of ES cells in the absence of DF (not illustrated). (C and D) Quantity of calcium and phosphate inthe colonies was enhanced with DF and TSH (1 mU/mL). (E) DF and TSH (0, 1, 5, and/or 10 mU/mL) enhanced the expression of (a) collagen type-1, (b) alkalinephosphatase (AP), and (c) osteocalcin (OC) at day 30 as assessed by qPCR.

Baliram et al. PNAS | September 27, 2011 | vol. 108 | no. 39 | 16279

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

Nov

embe

r 6,

202

0

Stem Cell Technologies) on gelatin-coated dishes in Dulbecco’s modified Eaglemedium, (Invitrogen Life Technologies, Inc.) supplemented with 10% FBS, 1%penicillin–streptomycin, and 1.5 × 104 M monothioglycerol (Sigma). Cultureswere maintained in a humidified chamber in a 5% CO2/air mixture at 37 ° C. EScell cultures were monitored daily, and the cells were passaged at 1:3 ratiosevery 2 d. For all experiments, cells were plated at the appropriate densitieswithout LIF for differentiation into its mesenchymal lineage, followed by os-teogenic differentiation factors (DFs). Lineage differentiation was accom-plished over 5 consecutive days, followed by osteogenic differentiation foranother 30 consecutive days with treatments containing differentiation (+)or no differentiation (−) factor with or without TSH. Media were changed

and treatments refreshed every 3 d. All osteoblast cultures were terminatedand analyzed on day 30 for the entire experiment except for CFU-fs, whichwas terminated and stained on day 12 of culture. All cultures were treatedwith osteogenic differentiation factors containing 10 mM β-glycerophosphate,1 mM dexamethazone, 50 μg/mL ascorbic acid, and 5 × 10−8 M vitaminD3. Vitamin D3, calciotrophic hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] was initally added on day 21 of culture.

CFU-f Staining for Alkaline Phosphatase. Cells were plated (1.0 × 106) in six-well plates (Falcon, BD Biosciences) and fixed with 10% formalin. At day 3,multicellular fibroblastoid colonies (CFU-fs), appeared that became in-

P<0.05

P<0.033

In

te

gra

te

d d

en

sity va

lu

es (A

.U

)

DF - + +TSH - - +

- + +- - +

- + +- - +

- + +- - +

OPG Wnt5a β-catenin β-actin

A B C D

E F G

Fig. 4. TSH stimulates noncannonical Wnt signaling. The effects of differentiation media (DF) and TSH (0, 5, or 10 mU/mL) on components of the Wnt-signaling pathway (assessed by real time PCR), namely Lrp5 (A), Lrp6 (B), β-catenin (C), Frizzled (Frz) (D), Wnt5a (E), and osteoprotegerin (OPG) (F) in 30-d EScell cultures. *P < 0.05 by ANOVA one-way analysis. (G) Densitometric values from Western blots showing the up-regulation of Wnt5a but not of β-catenin inthe cell lysates of cultures treated with DF+/TSH+. β-Actin is shown as the loading control.

P<0.05

P<0.05

A B C

Fig. 5. TSH stimulates protein kinase Cδ phosphorylation. (A) Differentiation medium (DF) (Materials and Methods) with and without TSH (0 and 1 mU/mL)triggered the phosphorylation of protein kinase Cδ (PKC-δ), but not of protein kinase A (PKA) as shown in this Western blot. β-Actin was the loading control.n = 2. (B and C) PKC-δ inhibitor, rotterlin (10 μM), inhibited the increase in type1 collagen and osteopontin (OPN) expression triggered by TSH (1 mU/mL)as assessed by qPCR.

16280 | www.pnas.org/cgi/doi/10.1073/pnas.1110286108 Baliram et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 6,

202

0

creasingly alkaline phosphatase positive over the following week. Uponfurther incubation, the colonies mineralized to form CFU-obs (1). CFU-fscolonies were stained for alkaline phosphatase using a kit as per the man-ufacturer’s recommendations (Sigma Aldrich) on day 12.

Staining for Mineralization of Osteoblasts. Mineralization of osteoblasts wasascertained by (i) von Kossa staining (28) and (ii) Alizarin red staining. Forvon Kossa staining, cells were plated and fixed as described above for al-kaline phosphatase staining. Cells were stained with 2% silver nitrate fol-lowed by 2.5% sodium thiosulfate (Sigma Aldrich). For Alizarin red staining,cells were similarly plated and fixed and stained with 2% Alizarin red stain(28) (pH 4.2) for 10 min, washed with dH2O several times, and finallyallowed to air dry at room temperature. Measurement of Alizarin red staineluted from these cells was used as an index of calcium release from oste-oblast cells in culture. Cells were grown in a 24-well dish with 1 × 105 cells/well, the stain was eluted by shaking in 100% iso-propanol for 10 min, andthe OD was measured at 540 nm.

Measurement of Calcium and Phosphorous in Osteoblasts. Cells were culturedin a 24-well dish at a density of 1 × 105 cell/well and allowed to differentiate.On day 30, cells were washed twice with 1× PBS, and 0.1 N NaOH was addedto each well for 24 h. Cells were then washed twice again with 1× PBS andcentrifuged, and the supernatant was collected and tested for calcium andphosphorous, as per the manufacturer’s instructions (Sigma Aldrich kit).

Detection of TSH Receptors in Osteoblasts by Flow Cytometry. Briefly, cellswere plated at a density of 6 × 106 cell/10-cm dish and then treated withosteogenic stimulation for 30 d. Cells were trypsinized, washed once withFACS buffer (PBS + 0.02% sodium azide), and then stained with TSH re-ceptor-specific antibody M1 to residues 381–385 (kindly supplied by B. ReesSmith, RSR Ltd., Cardiff, UK) for 1 h at room temperature. Following primaryantibody staining, the cells were washed with buffer and stained furtherwith an anti-mouse Fab PE-conjugated antibody (Jackson ImmunoResearch)at 1:200 dilution. Stained cells were then analyzed in the red channel (FL2)for PE staining. Negative controls stained with primary and secondary iso-types were also analyzed the same way.

RNA Isolation. Total RNA was isolated from cells using TRIZOL reagent(Invitrogen), and chromosomal DNA was removed in accordance with themanufacturer’s instructions. The RNA concentration was determined on thebasis of absorbance at 260 nm, and its purity was evaluated by the ratio ofabsorbance at 260:280 nm (>1.9). RNAs were kept frozen at −70 °C untilanalyzed. After digestion of genomic DNA by treatment with Ambion’sTurbo DNA-free DNase I (Ambion, Inc.), total RNA (1 μg) was reverse-tran-

scribed into cDNA with random hexamers using Advantage RT-for-PCR kit(Clontech Laboratories, Inc.).

Semiquantitative PCR. RT-PCRs for stem cell and osteoblast markers wasperformed using Titanium Taq polymerase (Clontech Laboratories). Details ofprimer sequences are shown in Table S1. Cycling conditions were as follows:94 °C for 1 min, followed by 30 cycles of amplification (94 °C denaturationfor 0.5 min; annealing for 1 min, annealing temperature dependent onprimers; 72 °C elongation for 2 min) with a final incubation at 72 °C for7 min. The amplified PCR products were separated on a 2% agarose gelsand visualized by ethidium bromide staining.

Quantitative RT-PCRs. Quantitative RT-PCRs (qRT-PCRs) were performed usingthe Applied Biosystems StepOne Plus real-time PCR system (Applied Bio-systems). The reactions were establishedwith a Power SYBRGreenmastermix(Applied Biosystems), 0.4 μL (2 μM) sense/antisense gene-specific primers,2 μL cDNA, and diethylpyrocarbonate-treated water to a final volume of 20μL. The PCR mix was denatured at 95 °C for 60 s before the first PCR cycle.The thermal cycle profile was the following: denaturizing for 30 s at 95 °C,annealing for 30 s at 57–60 °C (dependent on primers), and extension for60 s at 72 °C. A total of 40 PCR cycles were used. PCR efficiency, uniformity,and linear dynamic range of each qRT-PCR assay were assessed by the con-struction of standard curves using DNA standards. An average thresholdcycle from triple assays was used for further calculation. For each targetgene, the relative gene expression was normalized to that of the glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene usingApplied Biosystems Step One Plus Real-time PCR systems software. Datapresented (mean) are from three independent experiments in which allsample sets were analyzed in triplicate. The primers used are detailed inTable S1.

Western Blot Analyses. The cellular protein was extracted in the lysis buffer(pH 7.5) containing Tris·HCl (50 mM), Triton X-100 (0.5%), EDTA (2 mM), NaCl(150 mM), and 1 mM PMSF. The protein was measured by the method ofLowry (29): 50 μg of protein was electrophoresed in 12% SDS-polyacrylamidegel under reducing conditions and electrotransfered to nitrocellulosemembranes (Millipore). After blocking in 5% BSA, the membranes wereincubated with anti–β-catenin (1:2,000), anti-actin at a dilution of 1:2,000,anti-OPG, anti-Wnt5a at a dilution of 1: 500 (Santa Cruz Biotechnology),and anti–PKC-δ and anti-PKA antibodies at a dilution of 1:2,000 (Cell Sig-naling) and then probed with anti-rabbit, anti-goat, or anti-mouse second-ary antibody conjugated with peroxidase (Santa Cruz Biotechnology). Thesignals were detected with an ECL Western blotting detection system(Amersham Biosciences).

Osteoblast Signaling. Osteoblast cultures were grown in six-well plates ata density of 1 × 106 cells/well and cultured as described above (Cell Culture)with DFs and TSH (1 mU/mL). On day 5 or day 30, media were refreshed withserum-free media with or without rotterlin for 1 h before differentiationtreatment with or without TSH for another hour. Cultures were then ter-minated after the 2-h treatment.

Statistical Analyses. Differences resulting from treatments were assessed byStudent’s t-test or ANOVA. All differences were considered statistically sig-nificant if P < 0.05. All data are expressed as mean ± SEM. The statisticalanalyses were performed using Graph Pad v4.0.

ACKNOWLEDGMENTS. This work was supported in part by National Insti-tutes of Health Grants DK080459 (M.Z., L.S., and T.F.D.), DK069713 andDK052464 (T.F.D.), AG40132 and DK70526 (M.Z.), the David Owen SegalEndowment, and the VA Merit Review Program (T.F.D.).

1. Davies T, Marians R, Latif R (2002) The TSH receptor reveals itself. J Clin Invest 110:

161–164.2. Wang HC, Dragoo J, Zhou Q, Klein JR (2003) An intrinsic thyrotropin-mediated

pathway of TNF-alpha production by bone marrow cells. Blood 101:119–123.3. Inoue M, Tawata M, Yokomori N, Endo T, Onaya T (1998) Expression of thyrotropin

receptor on clonal osteoblast-like rat osteosarcoma cells. Thyroid 8:1059–1064.4. Abe E, et al. (2003) TSH is a negative regulator of skeletal remodeling. Cell 115:

151–162.5. Bauer DC, Ettinger B, Nevitt MC, Stone KL; Study of Osteoporotic Fractures Research

Group (2001) Risk for fracture in women with low serum levels of thyroid-stimulating

hormone. Ann Intern Med 134:561–568.

6. Martini G, et al. (2008) The effects of recombinant TSH on bone turnover markers and

serum osteoprotegerin and RANKL levels. Thyroid 18:455–460.7. Mazziotti G, et al. (2005) Recombinant human TSH modulates in vivo C-telopeptides

of type-1 collagen and bone alkaline phosphatase, but not osteoprotegerin pro-

duction in postmenopausal women monitored for differentiated thyroid carcinoma.

J Bone Miner Res 20:480–486.8. Zaidi M, et al. (2009) Thyroid-stimulating hormone, thyroid hormones, and bone loss.

Curr Osteoporos Rep 7:47–52.9. Zofkova I, Hill M (2008) Biochemical markers of bone remodeling correlate

negatively with circulating TSH in postmenopausal women. Endocr Regul 42:121–

127.

Fig. 6. TSH induces a local fast-forward loop for the regulation of boneremodeling. TSH stimulates Wnt5a secretion that enhances osteoblasto-genesis and mineralization, but also, through its action on frizzled (Frz),stimulates the production of osteoprotegerin (OPG). OPG, in turn, inhibitsbone resorption through its attenuation of RANKL signaling. These effectsunderscore the bone mass-enhancing effects of TSH that we have notedin vivo (13).

Baliram et al. PNAS | September 27, 2011 | vol. 108 | no. 39 | 16281

CELL

BIOLO

GY

Dow

nloa

ded

by g

uest

on

Nov

embe

r 6,

202

0

10. Iida K, Hino Y, Ohara T, Chihara K (2011) A case of myxedema coma caused by iso-

lated thyrotropin stimulating hormone deficiency and Hashimoto’s thyroiditis. EndocrJ 58:143–148.

11. Peeters RP, van der Deure WM, Visser TJ (2006) Genetic variation in thyroid hormone

pathway genes: Polymorphisms in the TSH receptor and the iodothyronine de-iodinases. Eur J Endocrinol 155:655–662.

12. Ma R, Morshed S, Latif R, Zaidi M, Davies TF (2011) The influence of thyroid-stimu-lating hormone and thyroid-stimulating hormone receptor antibodies on. Thyroid 21:

897–906.13. Hase H, et al. (2006) TNFalpha mediates the skeletal effects of thyroid-stimulating

hormone. Proc Natl Acad Sci USA 103:12849–12854.14. Sun L, et al. (2008) Intermittent recombinant TSH injections prevent ovariectomy-in-

duced bone loss. Proc Natl Acad Sci USA 105:4289–4294.15. Sampath TK, et al. (2007) Thyroid-stimulating hormone restores bone volume, mi-

croarchitecture, and strength in aged ovariectomized rats. J Bone Miner Res 22:849–859.

16. Lin RY, Kubo A, Keller GM, Davies TF (2003) Committing embryonic stem cells todifferentiate into thyrocyte-like cells in vitro. Endocrinology 144:2644–2649.

17. Tu X, et al. (2007) Noncanonical Wnt signaling through G protein-linked PKCdelta

activation promotes bone formation. Dev Cell 12:113–127.18. Thomas GP, Baker SU, Eisman JA, Gardiner EM (2001) Changing RANKL/OPG mRNA

expression in differentiating murine primary osteoblasts. J Endocrinol 170:451–460.19. Kubota T, Michigami T, Ozono K (2009) Wnt signaling in bone metabolism. J Bone

Miner Metab 27:265–271.

20. Baron R, Rawadi G (2007) Targeting the Wnt/beta-catenin pathway to regulate boneformation in the adult skeleton. Endocrinology 148:2635–2643.

21. Yadav VK, et al. (2008) Lrp5 controls bone formation by inhibiting serotonin synthesisin the duodenum. Cell 135:825–837.

22. Kato M, et al. (2002) Cbfa1-independent decrease in osteoblast proliferation, osteo-penia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wntcoreceptor. J Cell Biol 157:303–314.

23. Person AD, et al. (2010) WNT5A mutations in patients with autosomal dominantRobinow syndrome. Dev Dyn 239:327–337.

24. Yu HC, et al. (2010) Mechanical stretching induces osteoprotegerin in differentiatingC2C12 precursor cells through noncanonical Wnt pathways. J Bone Miner Res 25:1128–1137.

25. Yang D, Guo J, Divieti P, Bringhurst FR (2006) Parathyroid hormone activates PKC-delta and regulates osteoblastic differentiation via a PLC-independent pathway. Bone38:485–496.

26. Lima F, Niger C, Hebert C, Stains JP (2009) Connexin43 potentiates osteoblast re-sponsiveness to fibroblast growth factor 2 via a protein kinase C-delta/Runx2-de-pendent mechanism. Mol Biol Cell 20:2697–2708.

27. Vincent BH, et al. (2009) Bone marrow cells produce a novel TSHbeta splice variantthat is upregulated in the thyroid following systemic virus infection. Genes Immun 10:18–26.

28. zur Nieden NI, Kempka G, Ahr HJ (2003) In vitro differentiation of embryonic stemcells into mineralized osteoblasts. Differentiation 71:18–27.

29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with theFolin phenol reagent. J Biol Chem 193:265–275.

16282 | www.pnas.org/cgi/doi/10.1073/pnas.1110286108 Baliram et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 6,

202

0