A New Role for HERPUD1 and ERAD Activation in Osteoblast Differentiation and Mineralization

THE JOURNAL • RESEARCH • www.fasebj.org

A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization

† † † Luan Americo-Da-Silva,*´ ,1 Jheimmy Diaz,*,1 Mario Bustamante,*, Georthan Mancilla,*, Ingrid Oyarzun,*´ , † † Hugo E. Verdejo,*, and Clara Quiroga*, ,2 † *Division´ de Enfermedades Cardiovasculares, Facultad de Medicina, Pontificia Universidad Catolica´ de Chile, Santiago, Chile; and Advanced Center for Chronic Diseases, Universidad de Chile and Pontificia Universidad Catolica´ de Chile, Santiago, Chile

ABSTRACT: Bone integrity depends on a finely tuned balance between bone synthesis by osteoblasts and resorption by osteoclasts. The secretion capacity of mature osteoblasts requires strict control of proteostasis. Endoplasmic reticulum–associated degradation (ERAD) prevents the accumulation of unfolded ER proteins via dislocation to the cytosol and degradation by the proteasome. The ER membrane protein, homocysteine-inducible endoplasmic reticulum protein with ubiquitin-like domain 1 (HERPUD1), is a key component of the ERAD multiprotein complex which helps to stabilize the complex and facilitate the efficient degradation of unfolded proteins. HERPUD1 expression is strongly up-regulated by the unfolded protein response and cellular stress. The aim of the current study was to establish whether HERPUD1 and ERAD play roles in osteoblast differentiation and maturation. We evaluated preosteoblastic MC3T3-E1 cell and primary rat osteoblast differentiation by measuring calcium deposit levels, alkaline phosphatase activity, and runt-related transcription factor 2 and osterix expression. We found that ERAD and proteasomal degradation were activated and that HERPUD1 expression was increased as osteoblast differentiation progressed. The absence of HERPUD1 blocked osteoblast mineralization in vitro and significantly reduced alkaline phosphatase activity. In contrast, HERPUD1 overexpression activated the osteoblast differentiation program. Our results demonstrate that HERPUD1 and ERAD are important for the activation of the osteoblast maturation program and may be useful new targets for elucidating bone physiology.—Americo-Da-Silva,´ L., Diaz, J., Bustamante, M., Mancilla, G., Oyarzun,´ I., Verdejo, H. E., Quiroga, C. A new role for HERPUD1 and ERAD activation in osteoblast differentiation and mineralization. FASEB J. 32, 4681–4695 (2018). www.fasebj.org

KEY WORDS: osteoblastogenesis • bone • proteostasis • proteasome

Bone is a highly dynamic tissue, and its integrity depends Low BMD is associated with generalized bone fragility on a delicate balance between synthesis by osteoblasts and and an elevated risk of fracture in aging and in patholo- resorption by osteoclasts (1). The coordinated activity of gies, such as osteoporosis, osteogenesis imperfecta, and these cells is crucial for the maintenance of bone mineral cancer (2). The physiopathology of low BMD is related to density (BMD) as well as the structure, hardness, strength, gender and age, but also to such variables as systemic and characteristic metabolism of this tissue. inflammatory diseases, medication use, nutritional defi- ciencies, endocrine disorders, and other conditions that may promote decreases in osteoblast-dependent matrix ABBREVIATIONS: ALP, alkaline phosphatase; BiP, immunoglobulin heavy- secretion or increases in osteoclastic resorption (3, 4). chain-binding protein; BMD, bone mineral density; COL1, collagen type 1; Mature osteoblasts are those cells that are responsible ER, endoplasmic reticulum; ERAD, endoplasmic reticulum–associated degradation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HERPUD1, for bone formation and that specialize in large-scale se- homocysteine-inducible endoplasmic reticulum protein with ubiquitin- cretion of structural components of the bone matrix, col- like domain 1; OSX, osterix; PDI, protein disulfide isomerase; PERK, double- lagen type 1 (COL1) (5), and noncollagenous proteins, – stranded RNA-activated protein kinase like endoplasmic reticulum kinase; such as osteocalcin and osteoprotegerin (6). COL1 is POB, primary osteoblast; RUNX2, runt-related transcription factor 2; shHERPUD1, PLKO.1-shHERPUD1; shLUC, PLKO.1-shLUCIFERASE; mineralized by hydroxyapatite crystals to maintain bone shRNA, short hairpin RNA; UB, ubiquitin; UPR, unfolded protein response hardness and strength. Transitioning from preosteoblast 1 These authors contributed equally to this work. to mature osteoblast requires the activation of a genetic 2 Correspondence: Division´ de Enfermedades Cardiovasculares, Facultad program that is commanded by the master transcriptional de Medicina, Pontificia Universidad Catolica´ de Chile, Marcoleta 391, factors, runt-related transcription factor 2 (RUNX2) (7) and Santiago, Diagonal Paraguay 362, Santiago 8330077, Chile. E-mail: [email protected] osterix (OSX) (8). Secretory cells, such as osteoblasts, re- quire a finely tuned control to maintain proteostasis, in- doi: 10.1096/fj.201701229RR This article includes supplemental data. Please visit http://www.fasebj.org to cluding the regulation of protein biogenesis, folding, obtain this information. assembly, trafficking, and degradation (9).

0892-6638/18/0032-4681 © FASEB 4681 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Endoplasmic reticulum (ER) proteostasis, which is bone thickness, which are attributable to impaired osteo- crucial for the survival of secretory cells, is maintained by blast differentiation, a paucity of mature osteoblasts, and the unfolded protein response (UPR). This prosurvival abnormal ER retention of COL1 (22). Given this back- mechanism is activated when the demands of folding ground, protein traffic, secretion, and quality control overcome ER capacity. UPR involves a transient attenua- provide interesting new areas of study in osteoblast mat- tion of global protein synthesis, increased foldase and uration and bone mineralization, where ERAD activity chaperone expression, expanded ER volume, and a and HERPUD1 function remain unexplored. strengthening of protein degradation mechanisms that The objective of the current study was to determine are crucial for the quality control of secreted proteins whether HERPUD1 expression and ERAD regulation play (10). ER quality control is mediated by the activation of roles in osteoblast differentiation and maturation. Our ER-associated protein degradation (ERAD) and auto- results demonstrate that HERPUD1 expression is in- phagy, which are dependent on the proteasomal and creased during osteoblast differentiation in parallel with lysosomal machinery, respectively (10). increased ERAD and global proteasome activity. Absence ERAD is the most important pathway for the re- of HERPUD1 blocked osteoblast mineralization in vitro moval of misfolded ER proteins. Terminally unfolded and significantly reduced alkaline phosphatase (ALP) ac- proteins are recognized by chaperones and lectins, ret- tivity and bone differentiation marker expression. Of in- rotranslocated from the ER lumen to the cytoplasm, terest, HERPUD1 overexpression in preosteoblastic cells ubiquitinated, and degraded by the proteasome (11). was sufficient to activate the entire osteoblast differentia- Homocysteine-inducible ER protein with ubiquitin-like tion program. Our results reveal HERPUD1 and ERAD to domain 1 (HERPUD1), a recognized target of UPR, is a be new players in bone physiopathology and plausible component and regulator of ERAD (12). This integral therapeutic targets in low-BMD disorders. ER membrane protein is strongly up-regulated during ER stress (13, 14), then is quickly degraded after the restoration of ER homeostasis (15). HERPUD1 is ubiqui- MATERIALS AND METHODS tously expressed, but its expression is significantly higher in specialized secretory tissues, such as the pancreas (14). Reagents Expression levels in other specialized secretory tissues, a such as bone, remain unknown. HERPUD1knockout mice Fetal bovine serum, Opti-MEM, Trypsin, -minimum essential are viable, but demonstrate impaired glucose tolerance medium, penicillin/streptomycin, Lipofectamine 3000, Hoechst, Trizol, M-MLV Reverse Transcriptase, and Alexa Fluor Abs were without impaired insulin response during early develop- obtained from Thermo Fisher Scientific (Waltham, MA, USA). ment (16). Moreover, despite relatively low expression in Anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) the brain, HERPUD1 contributes to an adaptive cellular and anti–b-tubulin Abs were purchased from MilliporeSigma response in this organ that protects neural cells against (St. Louis, MO, USA). Bradford solution and PVDF membranes apoptosis both in vitro (17) and in vivo (16). were from Bio-Rad (Hercules, CA, USA). Secondary Abs and HERPUD1 is considered to be the master organizer of MG132 were obtained from Calbiochem (Burlington, ON, Canada). Anti-HERPUD1, anti-immunoglobulin heavy-chain-binding the ERAD machinery and interacts with E3-ubiquitin li- protein (BiP), ALP activity kit, Phosphatase Inhibitor Cocktail gase HRD1 (18, 19), ubiquitins (19), SEL1L, OS-9, and IV, and protein inhibitor were purchased from Abcam (Cam- derlin-1 (18) to degrade multiple proteins, such as CD3d bridge, MA, USA). Tunicamycin, thapsigargin, and puromycin and a1-antitrypsin Null Hong-Kong (19, 20). HERPUD1 were purchased from Biomol Research Laboratories (Plymouth was recently described as a modulator of ER-derived Meeting, PA, USA). Anti–protein disulfide isomerase (PDI) and quality control compartment assembly, where ERAD anti-ubiquitin (UB) Abs were from Cell Signaling Technology machinery and substrates are recruited for efficient deg- (Danvers, MA, USA). Fluorescence mounting medium was obtained from Dako (Carpinteria, CA,USA). CytoBuster Reagent radation (12). Moreover, we have reported that when was purchased from Merck (Darmstadt, Germany). Organic and HERPUD1 knockdown cells are subjected to stress, they inorganic compounds, acids, and solvents were acquired from not only demonstrate diminished proteasomal and ERAD Merck. Westar Supernova substrate was obtained from Cyanagen degradation activity, but also promote the degradation of (Bologna, Italy). Brilliant III Ultra-Fast SYBR Green QPCR Master polyubiquitinated aggregates via increased autophagic Mix was purchased from Agilent Technologies (Santa Clara, CA, flux (21). This finding suggests that, beyond its function as USA). Five clones of PLKO.1-shHERPUD1 (shHERPUD1) were a proteasome regulator, HERPUD1 could be crucial for obtained from lentiviral short hairpin RNA (shRNA) libraries that were developed by the Dana-Farber Cancer Institute (23, controlling global proteostasis. 24) and purchased from MilliporeSigma. MC3T3-E1 subclone 4 There is growing evidence that UPR components plays (CRL-2593) cells were chosen because of their high minerali- roles in bone turnover and the regulation of HERPUD1 zation capacity (25) and purchased from the American Type expression. In humans, mutations in PERK (double- Culture Collection (Manassas, VA, USA). stranded RNA-activated protein kinase–like endoplasmic reticulum kinase) —an ER stress sensor—may induce the rare autosomal recessive disorder, Wolcott-Rallison Cell lines, cultures, and differentiation conditions syndrome. Clinical features of Wolcott-Rallison syndrome MC3T3-E1 and HEK293T were cultured in growth medium that include early-onset (even neonatal) diabetes, multiple contained a-minimum essential medium without ascorbic acid epiphyseal dysplasia, osteoporosis, and growth retarda- and supplemented with 10% v/v fetal bovine serum and 100 U/ml tion (22). PERK loss-of-function mutations in mice result penicillin G sodium, 100 mg/ml streptomycin sulfate, and – in severe osteopenia and the loss of cortical and trabecular 250 ng/ml amphotericin B at 37°C with 5% CO2 95% air. To

4682 Vol. 32 September 2018 The FASEB Journal x www.fasebj.org AMERICO-DA-SILVA´ ET AL. Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. induce osteoblastogenesis, MC3T3-E1 cells were cultured in gels, then electrotransferred to PVDF membranes. After block- differentiation medium that contained 50 mg/ml ascorbic ing, primary Abs, anti-HERPUD1, anti-UB, anti-BiP, anti-PDI, acid, 10 nM dexamethasone, and 10 mM b-glycerol phosphate and anti-GAPDH, were incubated overnight at 4°C, and the for 3, 7, 14, and 21 d, taking care to only use cells at passage 26 as appropriate horseradish peroxidase–conjugated secondary Abs described by Yan et al. (26). We tested cells periodically to avoid were added. Membranes wereincubated with Westar Supernova the presence of mycoplasma. (Cyanagen, Bologna, Italy). Luminescence was visualized and digitalized by using a C-DiGit Blot Scanner (Li-Cor Biosciences, Lincoln, NE, USA), quantified with Image Studio Lite Software Isolation and culture of primary rat osteoblasts (v.5.2; Li-Cor), and normalized to GAPDH.

Primary osteoblasts (POBs) were obtained from 2-d-old neonatal Sprague-Dawley rat calvarial bones by using the pro- Real-time RT-PCR tocol described by Jonason and O’Keefe (27). In brief, excised and cleaned calvaria were subjected to sequential enzyme Total RNA was extracted from cultured MC3T3-E1 cells and digestion with 1 mg/ml of collagenase II. Cells were resus- POBs by using the Trizol reagent. Reverse transcription was pended in growth medium, maintained at 37°C in a humidi- performed by using M-MLV reverse transcriptase. cDNA was – – fied atmosphere of 5% CO2 95% air, cultured for 2 3duntil amplified with Brilliant III SYBR quantitative PCR Master Mix reaching 80–90% confluence, and plated according to experi- using specific primers for Herpud1, Col1a1, Sp7/Osx, RunX2, mental necessities. Alp, Ocn,andGapdh designed specifically for mice and rats. The Pfaffl comparative Ct method was used to analyze data. Values for each gene were normalized to Gapdh expression Alizarin red assay levels. Sequences of the primers used for RT-PCR are listed in Supplemental Table 1. To assess mineralized nodule formation and calcium deposits, POB and MC3T3-E1 cells were incubated with differentiation medium for 3, 7, 14, or 21 d. Cells were washed with PBS, fixed Flow cytometry with ice-cold methanol, washed, and then stained with 40 mM Alizarin Red S (pH 4.2) for 20 min at room temperature (25°C). Analysis of green fluorescent protein (GFP)m and CD3d-yellow Cell monolayers were subsequently washed with double- fluorescent protein (YFP) fluorescence were used to determine distilled H2O.SampleswereobservedwithanAxioImager proteasomal andERADactivity, respectively. Wild-type, shLUC, 3 Microscope (Carl Zeiss, Oberkochen, Germany) using a 4ob- or shHERPUD1 MC3T3-E1 cells were transiently transfected, jective. Images were recorded with AxioVision software (v.4.8; and fluorescence was quantified by flow cytometry in single live Carl Zeiss). Isopropanol was added to each well to dissolve the cells by using the FACSCanto system (Becton Dickinson, San dye from mineralized nodules. Absorbance values in each well Jose, CA, USA). Transfection rate was normalized to parallel the were measured at 620 nm in a Synergy 2 Multi-Mode Plate cell transduction of GFP. Reader (BioTek, Winooski, VT, USA).

Immunofluorescence, colocalization, and ALP activity radial analysis

POBs and MC3T3-E1 cells were maintained in differentiation Cells were fixed in paraformaldehyde (4% w/v), incubated medium for 3 or 7 d of differentiation. Alkaline phosphatase overnight at 4°C with anti-HERPUD1 and anti-PDI, and (ALP) enzymatic activity was measured with a colorimetric ALP – ’ revealed with Alexa Fluor conjugated Abs. For immuno- assay kit according to the manufacturer s protocol and read in a stained cells, nuclei were labeled with 10 mg/ml Hoechst Synergy 2 Reader. ALP activity (units/ml) was normalized for 33342 (Life Technologies, Carlsbad, CA, USA). We performed total protein content by using a standard Bradford solution. fluorescence microscopy by using a Zeiss LSM 510 confocal microscope (Carl Zeiss) that was equipped with a 363 objective. For each independent experiment, 5–10 cells at a time Lentivirus and HERPUD1 knockdown were imaged until at least 80 cells had been analyzed. Cells cell production were analyzed individually, then averaged. PDI and HER- PUD1 images were processed and analyzed by using ImageJ HEK293T cells were transfected in Opti-MEM medium with software (National Institutes of Healthy, Bethesda, MD, ’ Lipofectamine 3000 according to the manufacturer s instructions USA). Images were deconvolved by using the Iterative and using the following plasmids: VSV-G, pMDL, REV, and Deconvolution plugin (Developed by B. Dougherty, OptiNav, shHERPUD1 or PLKO.1-shLUCIFERASE (shLUC). After 48 h, Bellevue, WA, USA) and the background subtracted. Coloc- conditioned medium with lentiviral particles was collected and alization was quantified as the Manders’ coefficient by using m filtered through 0.45- m pore diameter filters. POBs and MC3T3- the JACoP plugin [Developed by Fabrice P. Cordelieres E1cells were incubated with conditioned medium, and cells were (French National Centre for Scientific Research, Paris, France) m selected with 4 g/ml puromycin after 48 h of viral transduction. and Susanne Bolte (Pierre and Marie Curie University-Paris 6, shHERPUD1- and shLUC-selected cells were differentiated. Paris, France)] for the fraction of HERPUD1 pixels also labeled as rough ER structure (PDI). We performed radial analysis of fluorescence as described in Bravo et al. (28). Each cell was Western blot analyzed individually according to size. By using the center of the nucleus as the starting point, 5 regions were defined: nu- For Western blot analysis, cells were washed and lysed with a clear, perinuclear, medial, radial (including the cell radius), CytoBuster protein extraction reagent that was supplemented and exterior. Nuclear and exterior regions were excluded with phosphatase and protease inhibitor cocktails. Total protein from the analysis as they were not relevant to the study. In the concentration was determined by Bradford assay. Equal remaining 3 regions, fluorescence, colocalization, or ratios amounts of protein were loaded on 8–15% SDS polyacrylamide were analyzed according to the experiment.

HERPUD1 AND ERAD DURING OSTEOBLASTOGENESIS 4683 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Statistical analysis The assay did not allow us to establish whether the increased proteasome activity was sustained for 7 or Data are expressed as means 6 SEM of at least 3 independent 14 d of differentiation, as total florescence dipped sharply ’ experiments. ANOVA and Student s t tests were carried out by to levels that were undetectable by flow cytometry as using GraphPad Prism7 (GraphPad Software, La Jolla, CA, maturation progressed. Therefore, to complement the USA). Comparisons between groups were carried out by using Tukey’s t test. above results, we evaluated proteasome efficiency during maturation by assessing polyubiquitinated protein accumulation at 0 and 7 d of differentiation. We observed that RESULTS polyubiquitinated protein accumulation was evident only in the presence of the general proteasome inhibitor, UPR is activated during MG132, with higher levels in differentiated vs. undiffer- osteoblastogenesis induction entiated cells (Fig. 2B). This finding suggests that more polyubiquitinated proteins are produced throughout the The presence of ascorbic acid (50 mg/ml), b-glycerol maturation process and that the proteasome is highly ef- phosphate (10 mM), and dexamethasone (10 nM) in the ficient its removal of polyubiquitinated proteins during culture medium is a potent inducer of osteoblast differ- osteoblast differentiation. entiation in vitro (26). We used these substances to elucidate the underlying protein quality control mechanisms of the osteoblast differentiation system, with a focus on Expression and distribution of HERPUD1 HERPUD1 and ERAD activation. Mouse MC3T3-E1 cells during osteoblastogenesis and POBs were subjected to differentiation conditions for 3, 7, 14, or 21 d. In vitro matrix mineralization was evalu- Givenpreviousfindingsthatdemonstratedhigh ated by Alizarin Red staining. A manifest increase in cal- ERAD activity in mature osteoblasts and the important cium deposits or mineralization was evident in both role of HERPUD1 in ERAD, we performed experi- MC3T3-E1 cells (Fig. 1A)andPOBs(Fig.1B), which was ments to elucidate the potential role of HERPUD1 in confirmed by quantification with Alizarin Red staining osteoblastogenesis. MC3T3-E1 cells and POBs were (Fig. 1C, E). Significantly elevated ALP activity in MC3T3- subjected to differentiation conditions for different E1 cells and POBs after 7 d of differentiation (Fig. 1D, F) times, and HERPUD1 levels were assessed. Western and increased genetic marker expression (Fig. 1G)were blot analysis showed that HERPUD1 levels increased also consistent with the findings of increased mineraliza- progressively from 6 h after the onset of differentiation A tion. As expected, our results also demonstrated increased (Fig. 3 ). This increase was significant at 24 h (Fig. 3A), expression of RUNX2 and OSX, the master regulators of with levels that were nearly 5-fold higher than those differentiation, as well as elevated ALP, COL1, and in undifferentiated control cells. HERPUD1 content osteocalcin expression (Fig. 1G). We also observed in- continued to rise over the longer term, with levels that creased UPR activation and induction of the chaperones were up to 70-fold higher than those in control cells and ER stress markers, BiP and PDI, during osteoclast by d 3 of differentiation in MC3T3-E1 cells. At 7 and maturation, which suggests increased protein-folding de- 14 d after the onset of differentiation, HERPUD1 pro- mand on the ER (Fig. 1H). tein content began to fall, although levels remained ;20-fold higher than those in undifferentiated control cells (Fig. 3B). To evaluate whether this effect was Increased proteasome and ERAD activity transcriptional, we measured HERPUD1 mRNA levels during osteoblast differentiation during osteoblastogenesis with quantitative RT-PCR (Fig. 3B) and observed a significant 4-fold increase in To determine for the first time, to our knowledge, HERPUD1 mRNA levels (Fig. 3C). POBs demonstrated whether ERAD and the proteasome are active dur- a similar response to the differentiation conditions, ing osteoblast maturation, we evaluated proteasome although at a different magnitude than that of MC3T3- activity by using plasmid reporters. MC3T3-E1 cells E1 cells. After 3 and 7 d of differentiation, HERPUD1 were transiently transduced with GFPm or CD3d-YFP. levels showed significant increases of 86 and 66%, re- These proteins are degraded by the proteasome and spectively (Fig. 3D). their subcellular distribution indicates whether the To determine subcellular localization and potential proteasome is actively removing cytosolic or ER pro- changes in HERPUD1 during maturation, we performed teins. GFPm is homogenously distributed in the cyto- indirect immunofluorescence analyses 3 and 7 d after the plasm, whereas CD3d-YFP is located in the ER; onset of osteoblast differentiation (Fig. 4A). As expected, therefore, decreased GFP or YFP fluorescence indicates cell size increased as osteoblast maturation progressed (Fig. increased global or ERAD-dependent proteasome ac- 4B). In undifferentiated cells, 100% of the cells remained tivity, respectively (21). Flow cytometry indicated that between 1 and 6 3 103 mm2 (34.4% were 1–2 3 103 mm2; preosteoblasts that were subjected to differentiation 47.5% were 2–4 3103 mm2;and18.1%were4–6 3 103 mm2), conditions for 3 d demonstrated both increased global with an average size of 2.7 3 103 mm2. In contrast, 15% of and ERAD-dependent proteasome activity (Fig. 2A), differentiated cells increased in size, some reaching 10 3 which suggests that these processes are active during 103 mm2 (Fig. 4B); however, 85% of differentiated cells osteoblast maturation. remained nearly unchanged, with an average cell size of

4684 Vol. 32 September 2018 The FASEB Journal x www.fasebj.org AMERICO-DA-SILVA´ ET AL. Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 1. Effect of differentiation media on preosteoblastic cells. A, B) MC3T3-E1 cells (A) and POBs (B) were treated for 3, 7, 14, and 21 d with differentiation medium [50 mg/ml ascorbic acid (AA), 10 nM dexamethasone (DEX), and 10 mM b-glycerol phosphate (BGP)]. Mineralization was visualized with Alizarin Red staining. Scale bar, 300 mm. C, D) Calcium deposits stained in MC3T3-E1 cells were dissolved and quantified (C), and ALP activity was assessed under differentiation conditions for 3, 7, and 14 d (D). E, F) Calcium deposits stained in POB were equally dissolved and quantified (E), and ALP activity in POB was assessed under differentiation conditions for 3 and 7 d (F). G) RUNX2, OSX, ALP, COL1, and osteocalcin (OCN) mRNA levels were determined by quantitative RT-PCR in basal conditions and 3, 7, or 14 d postdifferentiation and quantified relative to GAPDH (continued on next page)

HERPUD1 AND ERAD DURING OSTEOBLASTOGENESIS 4685 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 2. ERAD and global proteasome activation during osteoblast maturation. A) MC3T3-E1 cells were transduced with GFPm and CD3d-YFP plasmids and subjected to differentiation conditions 48 h after transduction for 3 d. Fluorescence was detected by ﬂow cytometry. B) Polyubiquitinated (poly-UB) protein levels were determined by Western blot analysis in total protein extracts of cells that were differentiated for 7 d in the presence or absence of 1 mg/ml MG132 for the last 24 h and normalized to GAPDH protein levels. Data are expressed as means 6 SEM of 5 independent experiments. Statistical signiﬁcance was calculated by using 2- way ANOVA. **P , 0.01 vs. basal undifferentiated condition, ++P , 0.01 vs. 7 d of differentiation of NT (untreated) cells.

2.2 3 103 mm2. A more detailed analysis of the confocal and 1 POB and 2 shHERPUD1 MC3T3-E1 cell clones were images revealed that the subcellular distribution of selected. shHERPUD1 clon1 and clon2 MC3T3-E1 cells dis- HERPUD1 was also different in basal vs. differentiated played 58 and 32% reductions in HERPUD1 protein levels, conditions. To quantify this observation, we performed respectively, compared with shLUC MC3T3-E1 cells (Fig. a radial analysis on the confocal images, as described in 5A), whereas shHERPUD1 clon1 POB demonstrated 25% Wang et al. (25), to estimate the spatial distribution of reductions in HERPUD1 levels (Fig. 5B). shHERPUD1 clones HERPUD1. As in the previous experiment, 5 cellular were subjected to differentiation conditions (14 d for MC3T3- regions were defined. Only the perinuclear, medial, and E1 cells and 7 d for POBs), and mineralization was assessed radial regions were additionally analyzed given the aims via Alizarin Red staining (Fig. 5C, D). shHERPUD1 clone of the experiment. Our results demonstrated that at d 3 expression impaired mineralization in vitro in both MC3T3- and d 7 of osteoblast maturation, HERPUD1 distribution E1 cells and POBs compared with shLUC cells (Fig. 5E, F). was not limited to the perinuclear region, where the rough This finding indicates that reducing HERPUD1 levels was ER is mainly located, but was also found toward the cell sufficient to prevent calcification in conditions under which periphery. The presence of HERPUD1 in the medial and the process would normally occur. Likewise, ALP enzyme radial regions increased significantly after 3 d of differ- function was reduced by approximately one half in entiation and remained high through at least d 7 (Fig. 4C). shHERPUD1 vs. shLUC MC3T3-E1 cells after 7 d of differ- In addition, we determined that HERPUD1 colocalization entiation conditions (Fig. 5G). To understand the mecha- with the ER-resident, PDI, decreased at 3 and 7 d of dif- nisms that might explain this lack of mineralization in ferentiation (Fig. 4D, E). Analysis of the surface plot re- shHERPUD1 cells, we assessed osteogenic program activity vealed that HERPUD1 distribution was not limited to the in these cells. We found that RUNX2 gene expression tended rough ER, stained with the PDI signal, but was also present to increase with differentiation in control cells, as seen in Fig. in other regions of the cell, even under basal conditions. 1; however, knocking down HERPUD1 seemed to prevent this increase and keep Runx2 mRNA at basal levels (Fig. 5H). Inhibition of osteoblastogenesis and protein To determine whether reduced HERPUD1 levels might degradation in shHERPUD1 cells affect ERAD or global proteasome function, we also measured polyubiquitinated protein accumulation in To determine whether increased HERPUD1 levels might shHERPUD1 clon1 and clon2 MC3T3-E1 cells. Basal pol- play a functional role in osteoblast differentiation, we gen- yubiquitinated protein accumulation was higher in these erated HERPUD1 knockdown MC3T3-E1 cells and POBs, cells compared with shLUC cells, with no significant transducing specific shRNA constructs by using lentiviral changes after 7 d of differentiation (Fig. 6A, B). Global and vectors. We tested the efficiency of 5 shHERPUD1 constructs, ERAD-dependent proteasome activity—as assessed by

mRNA expression in MC3T3-E1 cells. H) PDI protein levels and BiP mRNA levels were determined by Western blot analysis and quantitative RT-PCR and normalized to GAPDH levels in MC3T3-E1 cells. Data are expressed as means 6 SEM of at least 4 independent experiments. Statistical signiﬁcance was calculated by using ANOVA, and group comparisons were performed by using Tukey’s t test. *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 vs. basal undifferentiated condition.

4686 Vol. 32 September 2018 The FASEB Journal x www.fasebj.org AMERICO-DA-SILVA´ ET AL. Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 3. HERPUD1 expression during osteoblast maturation. A, B) HERPUD1 protein levels were determined by Western blot analysis in total protein extracts of MC3T3-E1 cells that were subjected to short-term differentiation conditions: 6, 12, and 24 h (A); or or long-term differentiation conditions: 3, 7, and 14 d (B), and normalized to GAPDH levels. C) HERPUD1 mRNA levels were determined by quantitative RT-PCR in MC3T3-E1 cells in basal conditions and at 3 and 7 d postdifferentiation, normalized to GAPDH mRNA expression and relative to undifferentiated control cells. D) HERPUD1 protein levels were determined by Western blot analysis in total protein extracts of POBs that were subjected to differentiation conditions for 3 and 7 d and normalized to GAPDH levels. Graphs represent means 6 SEM of at least 4 independent experiments. Statistical signiﬁcance was calculated by using ANOVA, and group comparisons were performed by using Tukey’s t test. *P , 0.05, **P , 0.01 vs. 0 d of differentiation.

using the fluorescent reporters GFPm and CD3d-YFP— performed 48 h after, defined as d 0. HERPUD1 over- demonstrated that reducing HERPUD1 levels resulted in expression at d 0 was quantified by Western blot analysis greater GFP fluorescence, indicating decreased proteasome (Fig. 7A, C) and quantitative RT-PCR (Fig. 7B). HERPUD1 activity (Fig. 6C, left); however, YFP fluorescence did not protein levels were 2-fold higher in HERPUD1-GFP vs. differ from that of control shLUC levels. A possible de- GFP MC3T3-E1 cells (Fig. 7A), and this increased expres- crease in ERAD activity in shHERPUD1 cells remains to sion was sustained through at least d 7 (Fig. 7B), whereas be confirmed (Fig. 6C, right). an 80% increase in HERPUD1 was achieved in HERPUD1- GFP POBs (Fig. 7C). HERPUD1 overexpression and induction Cell mineralization was also evaluated, as above, via of osteoblastogenesis Alizarin Red staining, ALP activity, and osteogenetic markers. HERPUD1 overexpression significantly increased Finally, to corroborate the above results, we determined MC3T3-E1 cell mineralization in vitro under basal, but not whether HERPUD1 overexpression enhanced minerali- differentiation, conditions (Fig. 7D). In contrast, HERPUD1- zation parameters. MC3T3-E1 cells or POBs were transfected overexpressing POBs demonstrated increased mineraliza- with HERPUD1-GFP and GFP constructs. All assays were tion in vitro only after the onset of differentiation (Fig. 7E).

HERPUD1 AND ERAD DURING OSTEOBLASTOGENESIS 4687 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 4. Cellular distribution of HERPUD1 during osteoblast maturation. A) Confocal images of HERPUD1 distribution during osteoblast maturation at 3 and 7 d of differentiation. Scale bar, 20 mm. B) Graphs represent the proportion of cells of various sizes during maturation (cellular area in square micrometer). C) Localization of HERPUD1 in MC3T3-E1 cells within regions of the radial analysis: perinuclear, medial, and radial at 0, 3, and 7 d of maturation conditions. D) Confocal images of HERPUD1 and PDI colocalization. Scale bar, 10 mm. Magnification and 3-dimensional surface plot of selected areas (right). E) HERPUD1 and PDI colocalization in MC3T3-E1 cells during maturation was calculated locally as the Manders’ coefficient for total cell area by differentiation time. Statistical significance was calculated by using ANOVA. **P , 0.01, ***P , 0.001, ****P , 0.0001 vs. 0dof differentiation; ++++P , 0.0001 vs. 3 d of differentiation.

4688 Vol. 32 September 2018 The FASEB Journal x www.fasebj.org AMERICO-DA-SILVA´ ET AL. Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 5. Effect of HERPUD1 knockdown on mineralization and osteoblast differentiation. A, B) MC3T3-E1 cells (A) or POBs (B) were transduced with lentivirus overexpressing different clones of shRNAs against HERPUD1 and an shRNA against Luciferase as control. Forty-eight hours post-transduction, cells were selected with 5 mg/ml puromycin, and HERPUD1 levels were determined by Western blot analysis. Densitometry results for HERPUD1 were normalized to GAPDH levels, and the percentage of residual HERPUD1 levels is presented below the image. Blots are representative of at least 4 independent experiments. C, D) MC3T3-E1 cells (C) and POB (D) shHERPUD1 and shLUC were subjected to differentiation conditions for 14 and 7 d, respectively, and stained with Alizarin Red. Scale bar, 300 mm. E) Stained calcium deposits in MC3T3-E1 cells were quantiﬁed and normalized to undifferentiated wild-type cells. F) Stained calcium deposits in POBs were quantiﬁed and normalized to undifferentiated shLUC cells. G) ALP activity was evaluated at 7 d of differentiation in clones of shHERPUD1 and shLUC MC3T3-E1 cells. H) Total RNA (continued on next page)

HERPUD1 AND ERAD DURING OSTEOBLASTOGENESIS 4689 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 6. Effect of decreased HERPUD1 levels on proteasome activity in preosteoblastic cells. A) Polyubiquitinated (poly-UB) protein accumulation was determined by Western blot analysis in total protein extracts of shHERPUD1 and shLUC cells under basal conditions and 7 d postdifferentiation in the presence of 1 mg/ml MG132. Blots are representative of 4 independent experiments. B) Basal and postdifferentiation polyubiquitinated protein accumulation was quantified by using Image Studio Lite software and normalized to GAPDH protein levels. Basal accumulation of polyubiquitinated protein in shHERPUD1 and shLUC cells is presented in the graph below. C) shHERPUD1 and shLUC MC3T3-E1 cells were transduced with GFPm and CD3d-YFP plasmids, and fluorescence was detected at 48 h post-transduction by flow cytometry. Data are expressed as means 6 SEM of at least 3 independent experiments. Statistical significance was calculated by using 2-way ANOVA. *P , 0.05 vs. shLUC cells.

Maturation-dependent and -independent increases in many of which are characterized by low BMD, bone ALP activity were greater in HERPUD1-overexpressing fragility, and an elevated risk of fracture. BMD de- cells (Fig. 7F). In terms of the osteogenic markers, only OSX creases with age, especially in postmenopausal expression was increased in HERPUD1-GFP cells com- women, as a result of reduced synthesis and miner- pared with GFP cells (Fig. 7G). Changes in RUNX2 or alization of the COL1 matrix (29) and excessive bone COL1 expression were not observed in these cells. resorption (30). Finally, we assessed proteasome efficiency by measur- Mature osteoblasts are important modulators of bone ing polyubiquitinated protein accumulation. We found mineralization. These cells are responsible for the synthe- that HERPUD1 overexpression tended to reduce basal sis and secretion of COL1 and other proteins, such as ALP polyubiquitinated protein accumulation (Fig. 7H). and osteocalcin, which modulate the availability of inorganic phosphate (31) and bind to hydroxyapatite (32) DISCUSSION during bone formation, respectively. Protein secretion depends not only on synthesis, transcription, and trans- Imbalanced bone turnover and homeostasis are re- lation rates, but also on efficient folding, post-translational sponsible for various diseases and pathologic conditions, modification, and traffic through the secretory pathway.

was extracted from shHERPUD1 MC3T3-E1 cells at 7 d of differentiation, and quantitative RT-PCR analysis was performed. Data are expressed as Runx2 normalized to Gapdh expression and relative to undifferentiated control cells. Graphs represent means 6 SEM of 5 independent experiments. Statistical signiﬁcance was calculated by using 2-way ANOVA. **P , 0.01, ***P , 0.001, ****P , 0.0001 vs. 0 d of differentiation; +P , 0.05, ++P , 0.01, +++P , 0.001 vs. 7 d of differentiation in shLUC cells.

4690 Vol. 32 September 2018 The FASEB Journal x www.fasebj.org AMERICO-DA-SILVA´ ET AL. Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Figure 7. HERPUD1 overexpression increases osteoblast maturation parameters. A) MC3T3-E1 cells were transduced with HERPUD1-GFP and GFP constructs and lysed at 72 h post-transduction to determine HERPUD1 protein levels by Western blot analysis. HERPUD1 levels normalized to GAPDH are presented below the images. Blots are representative of 5 independent experiments. B) HERPUD1 mRNA levels were determined by quantitative RT-PCR at 0 and 7 d post-transduction and quantiﬁed relative to Gapdh mRNA expression. C) POBs were transduced with HERPUD1-GFP and GFP constructs and lysed at 72 h post- transduction. HERPUD1 levels normalized to GAPDH are presented below the images. Blots are representative of 4 independent experiments. D) HERPUD1-GFP and GFP cells were subjected to differentiation conditions for 7 d and stained by using Alizarin Red. Calcium deposits in stained cells were quantiﬁed and normalized to undifferentiated GFP cells. Scale bar, 300 mm. E) HERPUD1-GFP and GFP POBs were subjected to differentiation conditions for 7 d and stained by using Alizarin Red. Scale bar, 300 mm. F) ALP activity was evaluated at basal conditions and after 3 d of differentiation in HERPUD1-GFP and GFP MC3T3-E1 cells. G) Total RNA was extracted at 7 d of overexpression, and quantitative RT-PCR analysis was performed. Data are expressed (continued on next page)

HERPUD1 AND ERAD DURING OSTEOBLASTOGENESIS 4691 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. The first step of the secretion pathway occurs in the ER are important for basal and parathyroid hormone– lumen, where the nascent protein is folded. Many factors, dependent osteoblast proliferation. Moreover, it is well including ER chaperone content, ER calcium concentra- known that several transcription factors, such as RUNX2 tion, foldase activity, and efficient protein quality control, (38) and OSX (39), and molecules, such as bone mor- are crucial at this step. Proteins that have achieved proper phogenetic protein 2, which controls osteoblast differ- conformation continue through the secretory pathway, entiation and the formation of new bone, are targets of whereas misfolded and unfolded proteins are retained in proteasomal degradation (40). Consistent with these the ER and degraded by ERAD or autophagy (9). previous findings, preosteoblastic MC3T3-E1 cells with Any imbalance in ER homeostasis triggers ER stress. knocked down HERPUD1 expression (Fig. 5) reduced The appropriate response to stress makes the difference the proliferation rate, which made it difficult to generate between cell survival or cell death. Three transmembrane a stable shHERPUD1 cell line (data not shown). In con- ER stress sensors may initiate the UPR pathway: activat- trast, under differentiation conditions, cell proliferation ing transcription factor 6, inositol-requiring kinase 1, and was unchanged, but osteoblastic mineralization capacity PERK (33). In conjunction, these pathways are crucial was indeed impaired (Fig. 5). for coordinating the ER stress response, which acti- Increased proteasome activity and efficiency prevented vates the transcription of multiple UPR genes that encode polyubiquitinated protein accumulation during cell dif- transcriptional factors, chaperones, phosphatases, anti– ferentiation (Fig. 2), which could eventually be toxic for the reactive oxygen species, and ERAD-related proteins (34), osteoblast. Although there are no data available regarding such as HERPUD1 (35). the effect of polyubiquitinated protein accumulation in Although there is evidence that UPR sensors and sig- osteoblasts, we suspect that abnormal accumulation of naling pathways contribute to osteoblastogenesis (22, 29), this protein may be related to cell death and bone disease there has been no evidence, to date, that would demon- progression, as has been observed in other secretor cells, strate a role for ERAD and its regulators, such as HER- such as pancreatic b-cells, where ubiquitinated protein PUD1, in this process. aggregation is associated with diabetes and cell damage In the current study, cells from the preosteoblastic cell (41). line MC3T3-E1 and POBs that were isolated from calvaria Osteoblast maturation was dependent on HERPUD1 in were kept in a controlled environment to induce differ- vitro, as shRNAs against HERPUD1 blocked mineraliza- entiation and mineralization in vitro. We monitored the tion (Fig. 5), whereas HERPUD1 overexpression pro- activity of the protein quality control mechanism, ERAD, moted mineralization (Fig. 7). These results could be the and the levels of the protein, HERPUD1, during the consequence of a dependence on HERPUD1 for efficient maturation process. Our experimental approach effi- proteasome function (Fig. 6). HERPUD1 levels were as- ciently induced osteoblast differentiation and mineraliza- sociated with changes in proteasome activity, reducing tion in vitro (Fig. 1), which, as expected, resulted in an global proteasome activity in shHERPUD1 cells (Fig. 6C) increased protein folding demand, as demonstrated by and decreasing polyubiquitinated protein accumulation in shifts in levels of BiP and PDI (Fig. 1H). Our results, which HERPUD1-overexpressing cells (Fig. 7H). demonstrated increased global and ERAD-dependent ALP is an ectoenzyme that is expressed in mineralization- proteasome activity during differentiation (Fig. 2), pro- competent cells, such as osteoblasts, the activity of which vide, to our knowledge, the first direct evidence of this depends on optimal folding and localization. ALP acts activity during osteoblast maturation, strongly suggesting that proteasome activity could be related to mineralization on the outer surface of the plasma membrane, extracel- capacity and bone physiology in general. lular vesicles, or by directly binding to COL1. This en- Currently, knowledge of the role of proteasomes in os- zyme is responsible for tissue mineralization, supplying teoblast biology is limited. In osteosarcoma MG-63 and the calcification substrate, inorganic phosphate (42, 43). HOS cell lines, the inhibition of the proteasome with MG132 As ALP traffics via the secretory route to exert its bio- increased the susceptibility to cisplatin-induced apoptosis; logic function in the extracellular space, alterations in however, in the osteoblastic hFOB 1.19 cell line, MG132 did this route could explain the absence of mineralization not modify apoptosis rates (36), which demonstrates that in shHERPUD1 MC3T3-E1 cells (Fig. 5G)andthein- theroleoftheproteasomechangesduringdifferentstages creased mineralization—even in the absence of differ- of osteoblast biology and bone physiopathology. entiation conditions—of HERPUD1-overexpressing The proteasome seems to play different roles in MC3T3-E1 cells (Fig. 7). ALP expression in bone has the proliferation and differentiation of preosteoblastic been demonstrated be sufficient to trigger the calcifi- cells. Murray et al. (37) determined that proteolytic— cation of other tissues and cell cultures (44). In addition, chymotrypsin-like and trypsin-like—proteasome activities it is known that HERPUD1 is important for the secretion

as Runx2, Osx, and Col1a1 normalized to Gapdh expression and relative to nontransduced, wild-type MC3T3-E1 cells. H) Polyubiquitinated protein accumulation was determined by Western blot analysis in total protein extracts of MC3T3-E1 GFP– and HERPUD1-GFP–overexpressing cells 72 h post-transduction. Polyubiquitinated protein levels normalized to GAPDH are presented below the images. Data are presented as means 6 SEM of 3 independent experiments. Statistical signiﬁcance was calculated by using 2-way ANOVA. *P , 0.05, **P , 0.01, ***P , 0.001 vs. undifferentiated cells; ++P , 0.01, +++P , 0.001 vs. same condition in GFP cells.

4692 Vol. 32 September 2018 The FASEB Journal x www.fasebj.org AMERICO-DA-SILVA´ ET AL. Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. of such proteins as insulin (45); therefore, changes in retention of nonmutated COL1 in the ER (29). Therefore, a HERPUD1 levels could be related to ALP secretion. similar phenomenon could explain the absence of miner- Another factor that could contribute to the in- alizationinHERPUD1knockdowncells.Additionalre- creased basal mineralization of HERPUD1-GFP cells search is needed to corroborate this idea. is elevated OSX expression (Fig. 7). This elevation Analyses of the role of HERPUD1 have been limited to may be attributable to a compensatory effect that is in its actions within the ER, but this protein may perform response to increased proteasomal degradation, as more complex functions throughout the cell. When we recent evidence demonstrates that OSX expression assessed the distribution of HERPUD1 during osteoblast is regulated by proteasome-dependent degradation maturation (Fig. 4), we observed that the protein was not (39). Additional studies are needed to confirm this restricted to the rough ER, where it colocalized with PDI concept. (Fig. 4C). The colocalization rate for HERPUD1 and PDI Conversely, it is possible that our results could be at- decreased during the course of MC3T3-E1 differentia- tributable to an unknown dependence of HERPUD1 on tion (Fig. 4D, E), which suggests that changes in the COL1 or noncollagenous protein traffic, secretion, or even distribution of this protein may influence activities in assembly. Currently, evidence of COL1 quality control is multiple compartments of the secretory route and implies restricted to the control and degradation of mutant COL1 an as-yet-unexplored role for HERPUD1 in these cells. To aggregates, which are removed by the proteasome or date, the only work to analyze changes in subcellular autophagy, depending on the protein mutation site (46, HERPUD1 distribution involved Golgi-to-ER relocaliza- 47). Moreover, in a murine model for osteogenesis tion, which was dependent on the E3-ubiquitin ligase, imperfecta, low BMD, fragility, and bone deformity are the POSH, and K63-linked ubiquitination to maintain calcium consequence of the intracellular retention of abnormal homeostasis (49). Future studies are needed to determine COL1 molecules and swelling of the ER cisternae (48). This whether HERPUD1 relocalizes to other organelles during scenario is similar to the osteogenesis imperfecta–like osteoblast maturation and to identify the function that 2 2 phenotype triggered in PERK / mice by the abnormal might mediate this process.

Figure 8. A new role for HERPUD1 during osteoblast differentiation. Activation of the osteogenic program in preosteoblastic cells induces an overload of ER folding demand and activates the UPR and its known target, the HERPUD1 protein. HERPUD1 expression controls the ERAD pathway and global proteasomal degradation, which prevents protein aggregation during cell maturation and promotes mineralization in vitro. HERPUD1 regulates protein quality control, and therefore proteostasis, during osteoblast differentiation, potentially acting as a regulator of bone turnover. AA, ascorbic acid; BGP, b-glycerol phosphate; DEX, dexamethasone; Pi, inorganic phosphate.

HERPUD1 AND ERAD DURING OSTEOBLASTOGENESIS 4693 Downloaded from www.fasebj.org by (191.125.51.54) on December 04, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()}, pp. 4681-4695. Finally, as summarized in Figure 8,ourresultsdem- 11. Vembar, S. S., and Brodsky, J. L. (2008) One step at a time: onstrate that HERPUD1, in its role as an ERAD regulator, endoplasmic reticulum-associated degradation. Nat. Rev. Mol. Cell Biol. 9,944–957 is crucial for activating the osteoblast maturation program 12. Leitman, J., Shenkman, M., Gofman, Y., Shtern, N. O., Ben-Tal, N., and mineralization in vitro through a mechanism that may Hendershot, L. M., and Lederkremer, G. Z. (2014) Herp coordinates be related to its subcellular relocalization, finely tuned compartmentalization and recruitment of HRD1 and misfolded – regulation of the secretory route, and proteasome activity. proteins for ERAD. Mol. Biol. Cell 25, 1050 1060 13. Hori, O., Ichinoda, F., Yamaguchi, A., Tamatani, T., Taniguchi, M., HERPUD1, therefore, represents a promising new in- Koyama, Y., Katayama, T., Tohyama, M., Stern, D. M., Ozawa, K., tervention target for bone physiopathology. Kitao, Y., and Ogawa, S. (2004) Role of Herp in the endoplasmic reticulum stress response. Genes Cells 9,457–469 14. Kokame, K., Agarwala, K. L., Kato, H., and Miyata, T. (2000) Herp, a ACKNOWLEDGMENTS new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J. Biol. Chem. 275, 32846–32853 15. Fumagalli, F., Noack, J., Bergmann, T. J., Cebollero, E., Pisoni, G. B., The authors acknowledge the Advanced Microscopy Facil- Fasana, E., Fregno, I., Galli, C., Loi, M., Solda,` T., D’Antuono, R., ı – ity, Unidad de Microscopá Avanzada Facultad de Medicina Raimondi, A., Jung, M., Melnyk, A., Schorr, S., Schreiber, A., (UMA–MED), Pontificia Universidad Catolica´ de Chile, and Simonelli, L., Varani, L., Wilson-Zbinden, C., Zerbe, O., Hofmann, Confocal Microscopy Unit, Faculty of Chemical and Pharma- K., Peter, M., Quadroni, M., Zimmermann, R., and Molinari, M. ceutical Sciences, Universidad de Chile, for equipment and (2016) Translocon component Sec62 acts in endoplasmic reticulum technical support, and Roberto Bravo-Sagua, Ph.D. [Instituto turnover during stress recovery. Nat. Cell Biol. 18, 1173–1184 de Nutricion´ y Tecnolog´ıa de los Alimentos (INTA), Uni- 16. Eura,Y.,Yanamoto,H.,Arai,Y.,Okuda,T.,Miyata,T.,andKokame,K. fi fi versidad de Chile, Santiago, Chile], for illustrations of our (2012) Derlin-1 de ciency is embryonic lethal, Derlin-3 de ciency fi results. This work was supported by the Comision´ Nacional de appears normal, and Herp de ciency is intolerant to glucose load and Investigacion Cient´ıfica y Tecnologica´ (Santiago, Chile) Grants ischemia in mice. PLoS One 7, e34298 ıfi 17. Chan, S. L., Fu, W., Zhang, P., Cheng, A., Lee, J., Kokame, K., and from Fondo Nacional de Desarrollo Cient´ co y Tecnologico´ Mattson, M. P. (2004) Herp stabilizes neuronal Ca2+ homeostasis and (FONDECYT) 11140470 (to C.Q.), 3160287 (to M.B.), 1150359 mitochondrial function during endoplasmic reticulum stress. J. Biol. (to H.V.), 11171024 (to J.D.), and Fondo de Financiamiento Chem. 279, 28733–28743 de Centros de Investigacion´ en Areas´ Prioritarias (FONDAP) 18. Schulze, A., Standera, S., Buerger, E., Kikkert, M., van Voorden, S., 15130011 (to H.V. and C.Q.). L.A.-D.-S. and J.D. are cofirst Wiertz, E., Koning, F., Kloetzel, P. M., and Seeger, M. (2005) The authors. The authors declare no conflicts of interest. ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway. J. Mol. Biol. 354,1021–1027 19. Kim, T. Y., Kim, E., Yoon, S. K., and Yoon, J. B. (2008) Herp enhances AUTHOR CONTRIBUTIONS ER-associated protein degradation by recruiting ubiquilins. Biochem. Biophys. Res. Commun. 369,741–746 L. Americo-Da-Silva,´ J. Diaz, M. Bustamante, G. Mancilla, 20. Kny, M., Standera, S., Hartmann-Petersen, R., Kloetzel, P. M., and and I. Oyarzún performed the research and participated Seeger, M. (2011) Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner. J. Biol. Chem. 286, in data analysis and discussion; H. E. Verdejo participated – in discussion; and C. 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