Essential Roles of Grp94 in Gut Homeostasis Via Chaperoning Canonical Wnt Pathway

Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway

Bei Liua, Matthew Staronb, Feng Honga, Bill X. Wua,c, Shaoli Sund, Crystal Moralesa, Craig E. Crossonc, Stephen Tomlinsona, Ingyu Kime, Dianqing Wue, and Zihai Lia,1

aDepartment of Microbiology and Immunology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425; bDepartment of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; cStorm Eye Institute, Medical University of South Carolina, Charleston, SC 29425; dDepartment of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425; and eDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520

Edited by Arthur L. Horwich, Yale University School of Medicine, New Haven, CT, and approved March 13, 2013 (received for review February 14, 2013) Increasing evidence points to a role for the protein quality control grp94 and Wnt pathway has not been reported. Furthermore, in the endoplasmic reticulum (ER) in maintaining intestinal homeo- a direct role for MesD or LRP5/6 in controlling adult intestinal stasis. However, the specific role for general ER chaperones in this homeostasis has not been demonstrated because of lack of ap- process remains unknown. Herein, we report that a major ER heat propriate genetic models. shock protein grp94 interacts with MesD, a critical chaperone for Here, we report that grp94 interacts with MesD, which is the Wnt coreceptor low-density lipoprotein receptor-related pro- necessary for proper cell surface expression of LRP6 and ca- tein 6 (LRP6). Without grp94, LRP6 fails to export from the ER to nonical Wnt signaling. Consistent with a role for grp94 in Wnt the cell surface, resulting in a profound loss of canonical Wnt sig- signaling, proliferation in intestinal crypts and intestinal integrity naling. The significance of this finding is demonstrated in vivo in in mice are critically dependent on grp94. that grp94 loss causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of in- Results testinal crypts, compromise of nuclear β-catenin translocation, loss grp94 Interacts with MesD and Chaperones LRP6. In an effort to of crypt-villus structure, and impaired barrier function. Taken to- expand the client network of grp94, we took an unbiased approach BIOLOGY gether, our work has uncovered the role of grp94 in chaperoning to immunoprecipitate grp94 clientale complex from a preleukemia LRP6-MesD in coordinating intestinal homeostasis, placing canoni- B-cell line, 14.GFP (9). We then resolved the complex on SDS/ DEVELOPMENTAL cal Wnt-signaling pathway under the direct regulation of the gen- PAGE and stained with Coomassie blue. We subjected the protein eral protein quality control machinery in the ER. bands that were specifically associated with grp94 pull-down to trypsin digestion, followed by tandem mass spectrometry (MS/MS) eat shock protein (HSP) grp94 (1), also known as gp96 (2), to identify grp94-associated proteins. We unexpectedly discovered Hencoded by HSP90b1 (3), is the endoplasmic reticulum (ER) MesD as a grp94-interacting molecule (Fig. 1A). MesD, so named paralog of cytosolic HSP90. Like other HSPs, grp94 is induced by after its critical role in mesoderm formation during early em- the accumulation of misfolded proteins (4) and binds and hydrol- bryogenesis, has been shown to be a critical chaperone for the fi izes ATP (5–7). As the most abundant protein in the ER lumen, it surface expression of LRP5/6 (22). To con rm the interaction is phylogenically conserved (8) and ubiquitously expressed in all between grp94 and MesD, we FLAG-tagged MesD and expressed nucleated cells. An important function of the ER is the steady-state MesD-FLAG in HEK293 cells, followed by a coimmunoprecipi- folding of nascent polypeptides into their mature tertiary/quater- tation study. Indeed, a strong interaction between the two was demonstrated (Fig. 1 B and C). Similarly, we found that grp94 nary structures and the assembly of large multimeric protein D complexes in the secretory pathway. Recent work demonstrates interacts with LRP6 (Fig. 1 ), which is a coreceptor for the cell that grp94 is the critical chaperone for multiple Toll-like receptors surface Wnt receptor Frizzled and is required for canonical Wnt (TLRs) and integrins (9–13) and that it participates in the unfolded signaling (23). After maturation and surface expression, LRP6 undergoes γ-secretase–dependent regulated intramembrane pro- protein response (UPR) (14). Without grp94, most integrins and teolysis (RIP) to liberate its extracellular and intracellular domain TLRs are unable to fold properly and, thus, fail to exit the ER and (24). Indeed, in wild-type (WT) mouse embryonic fibroblasts traffic to their proper “post-ER” compartment. Misfolded proteins (MEFs) (Fig. 1E), LRP6 undergoes RIP to release its extracel- are actively retained in the ER as a part of the ER quality-control lular domain (Fig. 1F). There are two forms of the full-length process, and their accumulation in the ER can result in ER stress LRP6 in WT cells: Endoglycosidase H (Endo H)-resistant surface and activation of the UPR. Up to 10% of cytosolic proteins are LRP6 and Endo H-sensitive LRP6 in the ER (Fig. 1F). However, dependent on HSP90 for folding (15); it is, therefore, unlikely that we found that in grp94 knockout (KO) cells, LRP6 does not un- grp94 clients are limited to TLRs and integrins. dergo RIP or transport to the cell surface, as indicated by the The intestinal epithelium is continually replenished through sensitivity of LRP6 to Endo H in grp94 KO cells (Fig. 1F). In the proliferation and differentiation of intestinal stem cells that further support of this conclusion, surface biotinylation followed reside within the intestinal crypts (16). Canonical Wnt signaling by avidin pull-down and immunoblot failed to detect LRP6 on the through the surface receptor, Frizzled, and its coreceptor low- surface of grp94 KO cells (Fig. 1G), although the same method density lipoprotein receptor-related protein 5 (LRP5) or LRP6, detected similar amounts of transferrin receptor and Wnt re- is instrumental for gut homeostasis (16). LRP5 and LRP6 are ceptor frizzled-4 (Fzd4) from WT and KO cells (Fig. 1 H and I). highly homologous to each other, and their function in the ca- Finally, we tested WT and grp94 KO cells for their abilities to nonical Wnt pathway is redundant in response to some Wnt ligands but not so to others (17–21). Mesoderm development (MesD), an ER chaperone, was recently shown to be necessary Author contributions: B.L., M.S., F.H., D.W., and Z.L. designed research; B.L., M.S., F.H., for surface expression of LRP5/6 and Wnt signaling (22). In- B.X.W., S.S., C.M., and I.K. performed research; B.L., M.S., F.H., B.X.W., S.S., C.M., C.E.C., terestingly, like grp94 and MesD, LRP5 and -6 are also necessary I.K., D.W., and Z.L. analyzed data; and B.L., M.S., S.T., and Z.L. wrote the paper. for mesoderm formation and gastrulation in mice (20), suggest- The authors declare no conflict of interest. ing a possible overlap among these proteins/pathways in early This article is a PNAS Direct Submission. development. However, the functional connection between 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1302933110 PNAS | April 23, 2013 | vol. 110 | no. 17 | 6877–6882 Downloaded by guest on September 25, 2021 a dose-dependent up-regulation of Axin2 mRNA in WT cells but not in KO cells (Fig. 1J). To determine the roles of grp94 in LRP6-MesD interaction, we expressed the both LRP6 and MesD in WT or grp94 knockdown HEK293 cells, followed by examination of the complex formation between the two. We found that knocking down grp94 resulted in a signiﬁcant reduction of LRP6-MesD complex (Fig. 2).

Conditional Deletion of grp94 Results in Loss of Intestinal Homeostasis and Reduced β-Catenin Nuclear Translocation. Intestinal homeostasis is critically dependent on the Wnt/β-catenin signaling pathway (25). For example, genetic deletion of β-catenin (26), LRP5/6 (27), and transcription factor 4 (Tcf4) (28) in mice results in profound loss of intestinal villus structure and death. If grp94 participates in Wnt coreceptor biogenesis, we would expect to see a similar intestinal phenotype in grp94 KO mice. We addressed this hypothesis using tamoxifen-inducible knockout strategy by fl fl crossing Hsp90b1 ox/ ox mice with a Rosa26ERT-cre mouse (29). Comparing with WT mice, we found that the KO mice experi- enced rapid weight loss, diarrhea, and hematochezia and ulti- mately death 12–14 d post-tamoxifen injection (PTI) in 100% of more than 200 mice studied (Fig. 3A). Gross pathological examination revealed significant intestinal dilatation, edema and hemorrhage, fecal obstruction, and thickening of the intestinal wall (Fig. 3B). Likely because of differences in the turnover rate of epithelium and the villus length (30), disease was often more pronounced in the terminal ileum, although the duodenum and jejunum were also affected to varying degrees without significant pathology in the colon. Like other Wnt-pathway KO mice, the pathology of the large bowel came later and was not easily observed because of death of the mice. The histopathological analysis revealed marked necrosis, neutrophil infiltration, and frank loss of intestinal villi and crypts in the ileum of grp94 KO mice (Fig. 3C). The pathology was apparent as further demonstrated by the pathology score to combine the degree of architectural loss and neutrophil infiltration (Fig. 3D). Importantly, loss of the gut epithelium is not limited to enterocytes. We observed near-total Fig. 1. grp94 interacts with MesD and is a critical molecular chaperone for loss of Paneth cells by lysozyme immunohistochemistry (Fig. 3E). LRP6. (A) Identification of MesD as a grp94-interacting protein. MS/MS analysis By BrdU pulsing, we found that there was significant reduction of of grp94-interacting proteins identified MesD. The sequence in red reflects BrdU uptake in the KO crypts (Fig. 3F), consistent with growth regions authenticated by MS. (B) Immunoblot of grp94 and MesD-FLAG fol- arrest and loss of β-catenin signaling (26). Remarkably, grp94 KO lowing immunoprecipitation of MesD-FLAG from HEK293 cell lysates. (C)Im- ileum had an almost complete loss of full-length LRP6 protein munoblot of grp94 and MesD-FLAG following immunoprecipitation of grp94 G fl from HEK293 cell lysates. Immunoprecipitation with isotype control antibody expression 12 d PTI (Fig. 3 ), likely re ecting the increased (ISO) is also shown. Expression of grp94 and MesD in whole cell lysate (WCL) is sensitivity of unfolded LRP6 to protease-rich environment in the indicated. Data are representative of two independent experiments. (D)Im- gut. The expression of E-cadherin, an important structural protein munoblot of LRP6 and grp94 following immunoprecipitation of grp94 from in the intestine (31), was not affected (Fig. 4 A and B). As ex- lysates of WT or grp94 KO MEFs. (E) Immunoblot analysis of grp94 from WT pected, ultrastructural analysis of the grp94-null ileum revealed the and grp94 KO MEF lysates. (F) Expression of endogenous LRP6 and its sensi- complete loss of microvilli and Paneth cells (Fig. 4C). The severe tivity to N-glycase Endo H and peptide N glycosidase F (PNGaseF) in WT and compromise of the gut integrity was also evident from the obser- β grp94 KO MEFs. Asterisk indicates cleaved LRP6. -Actin served as a loading vation of loss of gap junction protein ZO1 (Fig. 4D) and absence control. (G) WT or grp94 KO MEFs (duplicates) were subjected to surface of Goblet cells by Alcian blue stain (Fig. 4E). Consequently, biotinylation and pull-down with avidin beads, followed by immunoblot for cell surface LRP6. Total LRP6 was also blotted as a control. (H) Immunoblot of various proteins from the total lysates of WT and grp94 MEFs. After incubation of WT and grp94 mutant cells with EZ-Link Sulfo-NHS-SS-Biotin, lysates from cells were examined by immunoblot analysis for LRP6, transferrin receptor (Tfr), grp94, Fzd4, and tubulin. (I) Cell surface biotinylation of WT and grp94 KO MEFs. Lysates from cells treating with EZ-Link Sulfo-NHS-SS-Biotin were immunoprecipitated with NeutrAvidin beads and harvested. The pull-down– biotinylated membrane surface proteins were analyzed by immunoblotting. (J) Expression of Wnt target gene, Axin2, in response to Wnt stimulation. WT and grp94 KO MEFs were stimulated with Wnt-3a for 24 h, and fold expression of the Axin2 relative to unstimulated cells was measured by quantitative PCR. Data are representative of more than two independent experiments. Fig. 2. Knockdown of grp94 compromises LRP6-MesD interaction. LRP6-myc and MesD-FLAG were expressed in WT or grp94 knockdown (KD) HEK293 cells. Expression of indicated proteins in the whole-cell lysate of WT and KD respond to a known Wnt ligand, Wnt-3a. Axin2 is a negative cells was determined by immunoblot (IB). IP of LRP6-myc or MesD was then regulator of the Wnt-signaling pathway and is up-regulated in performed, followed by SDS/PAGE and IB for MesD-FLAG, grp94, and LRP6- response to Wnt ligand. As expected, Wnt-3a treatment led to myc. Two independent experiments were performed with similar findings.

6878 | www.pnas.org/cgi/doi/10.1073/pnas.1302933110 Liu et al. Downloaded by guest on September 25, 2021 development of the gut pathology, we reconstituted grp94 KO recipients with WT bone marrow (12). Over 90% chimerism of the gut-associated lymphoid tissues was confirmed by congenic marker (Fig. 6A). We found that WT hematopoietic system was unable to rescue the KO gut phenotype (Fig. 6B). Moreover, administration of broad-spectrum antibiotics, which effectively eliminated over 90% of the gut flora (33), failed to alter the severity or kinetics of the intestinal disease (Fig. 6C), suggesting that grp94 plays a critical and gut-intrinsic role in gut homeostasis via regulation of the canonical Wnt-signaling pathway. To fl fl further address this hypothesis, we crossed Hsp90b1 ox/ ox mice with a Villincre transgenic mouse (34) to allow for gut-specific deletion of grp94. Canonical Wnt pathway in the gut is dependent on the transcriptional activation complex between nuclear β-catenin and Tcf4, to transactivate downstream genes. Tcf4 plays indispensable roles in maintaining the crypt stem cells of the small intestine (28). Similar to Tcf4 KO mice, we found that gut-specific deletion of grp94 does not affect embryogenesis. The KO mice were born at expected Mendelian ratios (Fig. 6D). However, the intestinal epithelium-specific deletion of grp94 was associated with postnatal death of mice (Fig. 6D). The only 2 BIOLOGY

Fig. 3. Deletion of grp94 in mouse results in compromise of gut homeo- DEVELOPMENTAL stasis and loss of Wnt coreceptor LRP6. (A) Percent weight changes in WT and grp94 KO mice 12 d PTI. (B) Gross pathology showing bowel dilatation (∼three times of the diameter of the WT mice), obstruction, edema, and hemorrhage in the small intestine of grp94 KO mice (one representative mouse over more than 120 is shown). (C) H&E staining of gut sections of grp94 WT and grp94 KO mice 12 d PTI. The KO mice have fewer and shorter villi, especially in the ileum, which shows almost complete loss of villi with marked reduction of crypt. Multiple experiments (more than 30) were done with similar findings. (Scale bar: 100 μm.) (D) Quantification of gut pathology of KO (n = 5) and WT (n = 6) mice 12 d PTI. *P < 0.05. (E) Lysozyme stain of Paneth cells in the crypts of the ileum section. There was loss of crypt- villus structure and absence of Paneth cells in the KO ileum. (F) Immuno- fluorescence for BrdU and DAPI in the ileum of untreated (UT) and 12-d tamoxifen-treated mice. (Scale bar: 100 μm.) Comparing to the ileum of untreated mice, the tamoxifen-treated mice show significant loss of villi and crypts with markedly decreased BrdU uptake in the crypt. (G) Immunoblot for endogenous LRP6 at day 0 (WT mice) and day 12 (grp94 KO mice). grp94 and β-actin (loading control) expression is shown.

systemic dissemination of bacteria to the blood, liver, and mesenteric lymph node was evident with KO but not WT mice (Fig. 4F). To determine whether loss of canonical Wnt signaling is re- Fig. 4. Loss of intestinal barrier function and bacterial translocation in grp94 KO mice. (A) Normal expression of E-cadherin in ileum of grp94 KO mice. sponsible for the observed gut phenotype, we subjected grp94 KO fl β Immuno uorescence microscopy for E-cadherin (green) on day 0 (untreated) mice to treatment with glycogen synthase kinase 3 beta (GSK3 ) (WT) and day 12 (D12) grp94 KO mice. DAPI staining is shown (blue). (Scale inhibitor TWS119 (32). Inhibition of GSK3β is expected to lib- bar: 100 μm.) (B) Western blot analysis of E-cadherin in colon and ileum of WT erate β-catenin from its destruction complex and activate down- and KO mice. grp94 and β-actin (loading control) expression is shown. Data are stream Wnt target genes in a Wnt receptor/LRP6-independent representative of multiple mice per group and experiments. (C) Ultrastructural flox/wt ERT-cre manner. We found indeed that TWS119 treatment rescued the analysis of small intestine of grp94 KO mice. Hsp90b1 R26R (WT) and Hsp90b1flox/foxR26RERT-cre (KO) mice were treated for 12 d with tamoxi- gut pathology in KO mice, which correlated with increased crypt β fen. Distal ileum was dissected, carefully rinsed with PBS, and immediately cell proliferation (indexed by Ki67) and restored -catenin nu- fixed in 4% paraformaldehyde in PBS at 4 °C before processing for trans- clear translocation in the intestinal epithelial cells (Fig. 5A). mission electron microscope. LD, lipid droplets; MV, microvilli; P, Paneth cells. TWS119 treatment significantly improved the pathology score Loss of MV and Paneth cells can be readily seen. Data are representative of (Fig. 5B) and resulted in restoration of the Paneth cells in the four mice per group. (D) Loss of gap junction protein Z01 in the KO ileum by fl μ fi crypt (Fig. 5C). immuno uorescence. (Scale bar: 100 m.) (E) grp94 deletion leads to signi cant reduction of Goblet cells in all mice examined. (F) Presence of bacteria in cul- tured tissue lysate from mesenteric lymph nodes (MLN), liver (LIV), and blood Intestine-Specific Knockout of grp94 Recapitulates the Gut Pathology. (BLD) of grp94 KO mice. Representative images from each condition are shown. grp94 deletion in the hematopoietic system can cause gran- Numbers underneath these images represent average number of colonies after ulocytosis and defective lymphopoiesis (12). To rule out the overnight culture from six WT and five KO mice, followed by the frequency of possibility that KO immune system contribute indirectly to the mice with positive bacteria culture in parenthesis.

Liu et al. PNAS | April 23, 2013 | vol. 110 | no. 17 | 6879 Downloaded by guest on September 25, 2021 KO mouse now provides a unique experimental system to study the roles of LRPs in the biology of intestinal homeostasis in both physiological and pathological conditions. The proliferation and differentiation of the intestinal epithelium are tightly controlled by the Wnt/β-catenin pathway. The loss of enterocytes/villi and crypts in grp94 KO intestine closely resembled that of mouse models of gut-specific deletion of β-catenin (26) and LRP5/6 (27). Both β-catenin and LRP5/6 conditional KO mice developed severe intestinal pathology ∼4d after deletion. grp94 KO mice developed pathology around 10 d PTI, reflecting the requirement for 1 additional week to delete grp94. Our focus on LRP5/6 also came from two other obser- vations: that grp94 was found to complex with MesD and that LRP6 has some structural similarities to integrins, a known family of grp94-dependent client proteins (11). We found, indeed, that LRP6 expression in the intestine was significantly compromised in the absence of grp94. More importantly, LRP6 in grp94 KO cells is unable to acquire Endo H resistance and fails to export to cell surface, indicating that LRP6 is trapped in the ER in the absence of grp94. We also demonstrated that grp94 is required for optimal interaction between LRP6 and MesD. Further study is necessary to understand the precise

Fig. 5. β-Catenin activation rescues the intestinal pathology in grp94 KO mice. (A) After 10 d of treatment with DMSO or tamoxifen (TAM) with or without TWS119 (TWS), ileum tissues were stained for grp94, β-catenin, and Ki67 by immunohistochemistry. Shown also is an H&E-stained section. (Scale bar: 100 μm.) The marked ileum injury (mucosal ulceration, loss of villi, and decreased crypts) in TAM mice is reversed on H&E stain, and the immuno- histochemical staining pattern of indicated proteins are restored to that of DMSO-treated control mice with TWS119 treatment (TAM+TWS). (B) Quantiﬁcation of gut pathology of KO mice treated with indicated conditions (n = 5 per group). *P < 0.05. (C) Restoration of crypt-villus structure and appearance of Paneth cells (arrows) in the crypts of the ileum section of TAM-treated KO mice with TWS119.

survived mice out of 12 expected KO mice did so because of only partial knockdown of grp94. No mice with complete KO of grp94 survived. Examination of the newborn mice euthanized before death demonstrated the concurrent failure of β-catenin nuclear translocation (Fig. 6E). Histological analysis demonstrated decreased number of villi, significant reduction of cells in the crypt regions between the villi, decreased mitosis figures, and frequent “lifting” of the epithelial layer, which is reminiscent of Tcf4 KO mice (28). The complete phenocopy of gut-specific grp94 KO mice with Tcf4 KO mice led us to further conclude that the gut pathology associated with grp94 deletion is attributable to gut- intrinsic loss of Wnt signaling. Fig. 6. Gut-intrinsic roles of grp94 in gut homeostasis. (A) Flow-cytometric Discussion analysis of donor cells based on congenic marker CD45.2. (B)H&Estainingof ileum sections of WT→WT and WT→KO bone marrow chimeric mice 12 d PTI. Herein, we report the surprising and indispensable role for a major μ ER-resident molecular chaperone, grp94, in chaperoning LRP6 (Scale bar: 10 m.) (C) H&E staining of ileum sections of grp94 WT and KO mice treated with antibiotics before deletion of grp94 15 d PTD. Data are repre- and in intestinal homeostasis. grp94 binds to MesD and LRP6, and sentative of two independent experiments with multiple mice (n > 5) per it plays a critical role for the maturation and surface expression of group. (Scale bar: 25 μm.) (D) Loss of intestine-specific grp94 KO mice during LRP6. The essential roles of grp94 in LRP6 expression and the postnatal period. Villin-cre+/−Hsp90b1flox/WT mice were crossed with Villin- − − fl fl function in canonical Wnt pathway are established both in vitro cre / Hsp90b1 ox/ ox mice. The genotypes of newborn mice, as well as that of and in vivo. the postweaning adult mice, were determined. The observed frequency of + − fl fl We demonstrated that grp94 interact with both LRP6 and the Villin-cre / Hsp90b1 ox/ ox adult mice was significantly reduced from the “ ” expected value of 25%. *P < 0.05. (E) H&E and immunohistochemistry staining ER resident chaperone MesD (22). MesD does not appear to fl fl of grp94 and β-catenin of ileum sections from the villincreHsp90b1 ox/ ox and play any other role in ER function besides its unique function in cre flox/WT μ promoting LRP5/6 surface expression. Interestingly, grp94 also the control villin Hsp90b1 mice. (Scale bar: 25 m.) H&E stain of the ileum section of the KO newborn mice shows shorter villi with decreased cell appears to play an important role in mesoderm formation during number in intervillus crypt region (arrows) and mitosis, indicating less pro- development (35). Thus, our data strongly indicate that full LRP6 liferation activity. Immunohistochemistry stain reveals complete loss of grp94 maturation and cell surface expression is dependent on the co- expression in KO ileum with concurrent loss of β-catenin nuclear translocation ordinated action of both grp94 and MesD. The conditional grp94 compared with WT mice. A representative image from four mice is shown.

6880 | www.pnas.org/cgi/doi/10.1073/pnas.1302933110 Liu et al. Downloaded by guest on September 25, 2021 molecular mechanism of grp94-MesD-LRP interaction and its TWS119 (Cayman Chemical) was administered intraperitoneally after day 3 regulation during steady-state and pathological conditions. at 15 mg/kg body weight daily for 7 d (32). grp94 is an endoplasmic reticulum resident HSP and a master chaperone for TLRs and integrins (8–11, 13, 36). We also con- Antibiotic Treatment and Commensal Depletion. Mice were treated with 1 g/L sidered the possibility that loss of grp94-dependent client pro- ampicillin (Sigma), 1 g/L neomycin (Sigma), 1 g/L metronidazole (Sigma), and 0.5 g/L vancomycin (RPI Corp) in the drinking water for 1 mo. Commensal teins TLRs and/or integrins could partially contribute to the depletion was verified by fecal plating on LB agar without antibiotics (33). development of the intestinal pathology in grp94 KO mice. Water was changed every third day, and antibiotic treatment was continued However, mice that lack the TLR-signaling molecules myeloid throughout tamoxifen treatment. Tamoxifen was administered to mice at differentiation primary response 88 (MyD88) or TIR-domain- 5 μg/g body weight as above. Mice were killed at day 12, or their survival was containing adapter-inducing interferon beta (TRIF), although tracked indefinitely. Bacterial load from mice was quantified by inoculating more sensitive to experimental colitis, do not develop sponta- 300 μL of diluted blood (1:10 in PBS), homogenate of liver (50 mg/mL), or neous enteritis (37). Although loss of β1 integrin in the intestine mesenteric lymph node (25 mg/mL) to modified trypticase soy agar (TSA II) results in the development of colitis (38), β1 integrin expression plates with 5% (vol/vol) sheep blood (BD Biosciences), followed by overnight is not dependent on grp94 (12). Moreover, many of the integrin culture at 37 °C and counting of bacterial colonies. α-chains that pair with β1 are not dependent on grp94 either, α α Reverse Transcription and Quantitative PCR. Approximately 1-cm segments of with the exception of 2 (11). 2 knockout mice do not develop − gut disease, and although α2 is expressed in the mouse intestine, intestinal tissue were snap frozen and stored at 80 °C. RNA was extracted by fi α TRIzol (Gibco) method. cDNA was made by reverse-transcription PCR using the speci c function of 2 in the gut is unknown. Neither TLRs SuperScript polymerase II (Invitrogen). Quantitative PCR was performed us- nor grp94-dependent integrins have been directly linked to the ing Sybr Green (Applied Biosystems) method using the primer sets. Expres- gut homeostasis intrinsically. Most importantly, we demonstrated sion level was calculated using the formula 2−ΔCT and multiplied by a factor the complete phenocopy of gut-specific grp94 KO mice with Tcf4 of 10. β-Actin was used as an internal control. KO mice and that the gut phenotype associated with grp94 loss can be rescued by activation of canonical Wnt-signaling pathway Histopathology, Immunofluorescence, and Immunohistochemistry. Approxi- via GSK3β inhibitor. Thus, the pathogenesis of gut diseases in mately 1-cm segments of tissue were embedded in OCT freezing medium grp94 KO mice is primarily attributable to the loss of gut-in- (Thermo Scientific) and immediately frozen on dry ice or fixed in 4% trinsic canonical Wnt pathway, rather than the lack of other formaldehyde/PBS, rehydrated in 30% sucrose/PBS, before embedding in OCT medium. Samples were stored at −80 °C until sectioning. Five to 7-μmsec- functional clients of grp94. BIOLOGY tions were cut on a cryostat onto poly-L lysine-coated slides (Sigma). For

In summary, we have uncovered a function of a major ER lu- DEVELOPMENTAL histological examination, slides were immediately stained with hematoxylin/ minal HSP grp94 in chaperoning LRP6 and Wnt signaling. Given eosin (H&E). Gut pathology score is defined by summation of two parameter the importance for Wnt in tumorigenesis and other aspects of scores to achieve a range of 0–6: inflammation (0, no inflammation; 1, neu- development (25, 39), a previously unappreciated and critical role trophil rarely discernible; 2, presence of neutrophils in every high power for grp94 in the Wnt signaling may have fundamental implications field; 3, collection of >10 neutrophils in any given area); tissue destruction in linking the general ER protein homeostasis with Wnt signaling. (0, normal structure; 1, villus length shortened by 50%; 2, flattened mucosa Elucidation of the precise mechanism of grp94 in chaperoning with loss of villi; 3, evidence of submucosa ulceration). For staining Goblet LRP6 will also facilitate grp94-targeted therapeutics for cancer. cells, fixed tissue was stained with Alcian blue solution for 20 min at room temperature and then rinsed thoroughly in tap water, followed by coun- Methods terstain with Kernechtrot’s nuclear fast red solution for 5 min. After rinsing Mice and Genotyping. Conditional Hsp90b1-deficient mice were described in distilled water, the tissue was dehydrated and mounted. For immuno- fl fi previously (10, 11, 40). villincre mice were purchased from The Jackson Labo- uorescence, slides were xed in ice-cold acetone. Alternatively, for grp94 fi ratory. Animal use was approved by the Medical University of South Carolina intracellular staining, slides were xed in 4% formalin/PBS for 20 min and Animal Care Committee. permeabilized with ice-cold methanol for 20 min. Slides were washed in PBS; Fc receptor was blocked and/or slides were blocked in 10% goat serum/PBS Plasmids. grp94 constructs in MigR were described previously (10). Human for 30 min. Primary antibodies were diluted in 2% BSA/PBS, and slides were LRP6-myc was a generous gift from Cristof Neihrs (Deutsches Krebsfor- stained for 45 min. After washing in PBS, slides were stained with secondary shungszentrum, Heidelberg) and Yonghe Li (Southern Research Institute, antibody (or directly conjugated antibodies) diluted in 2% BSA/PBS for Birmingham, AL). Mouse MesD-FLAG and mouse LRP6-myc plasmids were 30 min. Slides were washed again and counterstained with nuclear stain, fi μ a generous gift from Janet Lighthouse and Bernadette Hoeldner (Stony DAPI. For BrdU staining, xed-tissue samples were cut into 10- m sections fi Brook University, Stony Brook, NY). Site-directed mutagenesis was per- and xed a second time in 10% formalin/PBS for 30 min. After washing, formed on parental grp94/MigR using a commercially available kit (Stra- slides were treated in 2 M HCl/H2O for 20 min at 37 °C. Slides were rinsed in tagene) and verified by sequencing. Transient transfection of HEK293 cells PBS and neutralized in two washes in 0.1 M sodium borate buffer (pH 8.5). was performed using 5–10 μg of plasmid DNA and Lipofectamine 2000. Cells After thorough washing, slides were stained with anti-BrdU antibody (MoBU-1 were analyzed 48 h after transfection. or ZBU-30; 1:25; Invitrogen).

Cell Lines. HEK293 cells were originally obtained from the ATCC. WT grp94 Electron Microscopy. Tissues were dissected and ﬁxed in 4% buffered para- and tamoxifen-inducible KO MEFs were generated and immortalized using formaldehyde, osmicated, stained in block with uranyl acetate, dehydrated, transduction of large-T antigen retrovirus. After cloning, MEF cells were either and embedded in Poly/Bed resin. Thick 1-μm sections were cut on a Leica EM treated with ethanol (vehicle) or 1 mM hydroxy-tamoxifen (Sigma-Aldrich) to UC7 ultramicrotome and examined in the light microscope. Thin 70- to generate grp94-null MEFs. MEF cells were maintained as WT or grp94 KO. 80-nm sections were cut and collected on 200 Cu/Rh mesh grids, stained with uranyl acetate and lead citrate, and observed in a Hitachi H-7650 trans- Antibodies. Most antibodies were purchased from Sigma-Aldrich except the mission electron microscope. antibodies against the following antigens: Lysozyme (Abcam), β-cat (Cell Signaling), grp94 (Enzo Life Sciences), Ki67 (Nova Biologicals), ZO1 (Abcam), BrdU Pulse. Mice were injected intraperitoneally with BrdU (1 mg) in PBS 24 h BrdU (Invitrogen), transferrin (Invitrogen), and LRP6 (Cell Signaling). before euthanasia.

Tamoxifen-Inducible Deletion of grp94 and Model of Acute Intestinal Disease Protein Extraction, Immunoprecipitation, and Western Blot. Protein extraction, and Bone Marrow Chimeras. grp94 KO mice and bone marrow chimeras were immunoprecipitation, and Western blot were carried out as described pre- described previously (12). Tamoxifen citrate (Sigma-Aldrich) was dissolved at viously (10). Briefly, HEK293 or MEF cells were harvested by trypsin-EDTA or 10 mg/mL in peanut or corn oil and further diluted to 1 mg/mL for injections. 5 μM EDTA, subjected to dithiobis (succinimidyl propionate) (Thermo Scientific) Low-dose tamoxifen (5 μg/g body weight) was injected intraperitoneally to for 30 min at room temperature, washed in PBS, and lysed on ice in radio- Hsp90b1flox/wtR26RERT-cre (WT), Hsp90b1flox/flox,orHsp90b1flox/foxR26RERT-cre immunoprecipitation assay (RIPA) lysis buffer plus protease inhibitor mixture (KO) mice for 0–12 consecutive days. For rescue experiment, GSK3β inhibitor (Sigma-Aldrich). Nuclear-free protein lysate was quantified by Bradford assay,

Liu et al. PNAS | April 23, 2013 | vol. 110 | no. 17 | 6881 Downloaded by guest on September 25, 2021 and an equal amount of lysate was incubated with antibodies or isotype proteins in NeutrAvidin beads were eluted with SDS sample buffer and sub- control as indicated. grp94 was immunoprecipitated using 9G10 antibody jected to Western blot analysis. (Stressgen) and Protein-G beads (Pierce), and MesD-FLAG was immunoprecipitated using anti-FLAG antibody (BioM2; Sigma) and Neutravidin beads Wnt Signaling. WT and KO MEF cells were seeded at 1 × 105 cells per well in fi (Thermo Scienti c). a 12-well plate the night before. The next day, cells were either stimulated with Wnt 3a (Peprotech) at the indicated dose or left unstimulated. Quan- – In-Gel Digestion and Nano LC-MS/MS Analysis of grp94-Associated Proteins. titative PCR for Axin2 was performed using the following primers: 5′- grp94 immunoprecipitates were resolved on SDS/PAGE. In-gel digestion of TGACTCTCCTTCCAGATCCCA-3′ (forward) and 5′-TGCCCACACTAGGCTGACA-3′ unique bands was performed using trypsin, followed by microcapillary HPLC (reverse). Relative fold expression was calculated by the ΔΔCT method relative and LC-MS/MS analysis on the linear trap quadrupole. The obtained MS/MS −ΔΔ to β-actin control using the following formula: 2 CT. data were subjected to database searches of mouse proteome using SEQUEST software, as described (41). ELISA. Serum was collected at the time of euthanasia. TNFα, IL-6, and IL-12p40 ’ Cell Surface Biotinylation. After washing cells three times with 1× PBS CM ELISAs (BD Bioscience) were performed according to the manufacturer s protocol. [10 mM potassium phosphate (pH 7.5), 140 mM NaCl, 0.1 mM CaCl2, and ’ χ2 1 mM MgCl2], cells were incubated for 30 min with the 1× PBS CM con- Statistics. Error bar represents standard deviation. The Student s t test or taining 0.5 mg/mL of EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific; test was used to determine whether the difference between two groups was catalog no. 21331) on ice with rocking. The biotinylation reactions were statistically significant (P < 0.05).

then incubated with 1× PBS CM containing 50 mM NH4Cl on ice for 5 min to quench free biotin. Then, the cells were washed three times with 1× PBS CM ACKNOWLEDGMENTS. We thank the past and present members of our and lysed in lysis buffer containing 1.25% Triton X-100, 0.25% SDS, 50 mM laboratories for their input throughout the course of this work. B.L. is a Tris·HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 5 mg/mL iodoacetamide, 10 μg/mL National Institutes of Health (NIH) KL2 scholar and is supported by the South APMSF, and protease inhibitor mixture (Roche). Whole-cell lysates were Carolina Clinical and Translational Research Institute at the Medical University of South Carolina (NIH Grants KL2RR029880 and UL1RR029882). The work was centrifuged at 15,000 × g at 4 °C for 15 min, and the supernatant was collected ﬁ also supported, in part, by the Flow Cytometry and Cell Sorting Shared and incubated with NeutrAvidin beads (Thermo Fisher Scienti c; catalog no. Resource, Hollings Cancer Center, Medical University of South Carolina (NIH 29200) at 4 °C overnight while rotating. The NeutrAvidin beads were centri- Grant P30 CA138313). Z.L., C.E.C., S.T., and D.W. are supported by NIH grants. fuged at 1,000 × g at 4 °C for 3 min and washed with wash buffer [0.5% Triton Z.L. is the Abney Chair Remembering Sally Abney Rose in Stem Cell Biology X-100, 0.1% SDS, 50 mM Tris·HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA). The and Therapy.

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