Bone 74 (2015) 29–36

Contents lists available at ScienceDirect

Bone

journal homepage: www.elsevier.com/locate/bone

Original Full Length Article Glucose-dependent insulinotropic polypeptide (GIP) directly affects fibril diameter and collagen cross-linking in osteoblast cultures

Aleksandra Mieczkowska a, Beatrice Bouvard b, Daniel Chappard a,c, Guillaume Mabilleau a,c,⁎ a GEROM Groupe Etudes Remodelage Osseux et bioMatériaux—LHEA, IRIS-IBS Institut de Biologie en Santé, CHU d'Angers, LUNAM Université, 49933 Angers Cedex, France b Service de Rhumatologie, CHU d'Angers, 49933 Angers Cedex, France c SCIAM, Service Commun d'Imagerie et Analyses Microscopiques, IRIS-IBS Institut de Biologie en Santé, CHU d'Angers, LUNAM Université, 49933 Angers Cedex, France article info abstract

Article history: Glucose-dependent insulinotropic polypeptide (GIP) is absolutely crucial in order to obtain optimal bone Received 4 October 2014 strength and collagen quality. However, as the GIPR is expressed in several tissues other than bone, it is difficult Revised 19 December 2014 to ascertain whether the observed modifications of collagen maturity, reported in animal studies, were due to di- Accepted 5 January 2015 rect effects on osteoblasts or indirect through regulation of signals originating from other tissues. The aims of the Available online 10 January 2015 present study were to investigate whether GIP can directly affect collagen biosynthesis and processing in osteo- Edited by Ms. J. Aubin blast cultures and to decipher which molecular pathways were necessary for such effects. MC3T3-E1 cells were cultured in the presence of GIP ranged between 10 and 100 pM. Collagen fibril diameter Keywords: was investigated by electron microscopy whilst collagen maturity was determined by Fourier transform infra- GIP red microspectroscopy (FTIRM). Osteoblast GIP treatment resulted in dose-dependent increases in lysyl activity and collagen maturity. Furthermore, Collagen GIP treatment shifted the collagen fiber diameter towards lower value but did not significantly affect collagen heterogeneity. GIP acted directly on osteoblasts by activating the adenylyl cyclase–cAMP pathway. This study provides evidences that GIP acts directly on osteoblasts and is capable of improving collagen maturity and fibril diameter. © 2015 Elsevier Inc. All rights reserved.

Introduction tissues other than bone, it is difficult to ascertain whether the observed modifications in collagen quality are due to direct effects on osteoblasts Glucose-dependent insulinotropic polypeptide (GIP) is an important or indirect through regulation of signals originating from other tissues. gastro-intestinal hormone synthesized and secreted into the blood Bone tissue is considered to be a two-component material made stream by intestinal endocrine K cells after ingestion of a mixed meal primarily of mineral (poorly crystalline hydroxyapatite) and organic [1–3]. In humans, fasting plasma GIP is ranged between 12 and 92 pM (collagen type I) matrices [13]. The mineral component confers stiffness and increases to 35–235 pM postprandially [4]. To induce a biological to the bone tissue [14,15] whilst collagen confers a degree of tensile response, GIP binds to a specific glucose-dependent insulinotropic strength, ductility and toughness [16,17]. Collagen type I biosynthesis polypeptide receptor (GIPR) expressed in several tissues including by osteoblasts is a complex process and includes intracellular post- bone tissue [5–9]. However, despite expression of its receptor at the sur- translational modifications, assembly of procollagen chains and secre- face of osteoblasts, little is known about the osseous effects of the GIP/ tion [18]. In the extracellular space, C-terminal and N-terminal GIPR pathway. Previously, we reported that the deletion of the Gipr propeptides are cleaved resulting in collagen fibril formation. The in mouse resulted in dramatic alterations of trabecular bone ultimate processing of collagen fibrils is represented by enzymatic microarchitecture [10]. We also evidenced that bone strength and ma- cross-linking. Cross-linking stabilizes the collagen network and is due terial properties were markedly reduced in this animal model with a to oxidative deamination of ε-amino groups of specificlysineand dramatic 16% decrease in collagen maturity [11]. Furthermore, treat- hydroxylysine residues by , forming aldehyde moieties ment of healthy Copenhagen rats with a stable GIP agonist resulted in [19]. These aldehydes are highly reactive and lead to formation of cova- a 13% increase in collagen maturity [12]. Taken together, these results lent crosslinks that are critically required for the formation of functional suggest that the GIP/GIPR pathway is involved in the control of collagen mature collagen. The assembly of collagen molecules into fibrils has quality in the bone matrix. However, as the GIPR is expressed in several been described as entropy driven, similar to that occurring in other self-assembling such as microtubules or actin filaments [20]. ⁎ Corresponding author. Fax: +33 244 688 451. However, data obtained from genetically-modified animals revealed E-mail address: [email protected] (G. Mabilleau). that several other molecules, present in the extracellular bone matrix,

http://dx.doi.org/10.1016/j.bone.2015.01.003 8756-3282/© 2015 Elsevier Inc. All rights reserved. 30 A. Mieczkowska et al. / Bone 74 (2015) 29–36 may regulate collagen deposition and fibril formation. Among them, cAMP determination -containing molecules such as and are suspected to play a major role in collagen deposition and fibril for- Cells were plated at a density of 3 × 105 cells/cm2 and grown for 48 h mation as demonstrated by abnormal collagen fibrils in deficient ani- in αMEM supplemented with 5% FBS, 5% bovine calf serum, 100 U/mL mals [21,22]. Furthermore, evidences have been provided that lysyl penicillin, and 100 μg/mL streptomycin in a humidified atmosphere oxidase activity is also a pre-requisite for normal collagen deposition enriched with 5% CO2 at 37 °C. Then cells were starved in αMEM supple- and fibril organization [23]. mented with 0.5% BSA for 16 h and incubated for 15 min in αMEM sup- The aims of the present study were to investigate whether GIP di- plemented with 0.5% bovine serum albumin and 1 mM IBMX prior to rectly affects collagen deposition and maturation in osteoblast cultures stimulation with GIP. After 45 min, cells were washed with ice-cold and to decipher what molecular pathways were necessary for such PBS, incubated in lysis buffer and centrifuged at 12,000 g for 10 min. Su- effects. pernatants were collected and stored at −80 °C until use. Cyclic AMP determination was performed with a fluorometric commercially avail- able kit (R&D Systems Europe, Abingdon, UK) according to the Material and methods manufacturer's recommendations.

Reagents Lysyl oxidase (LOX) activity

Alpha-Minimum Essential Medium Eagle (α-MEM), fetal bovine LOX activity was assessed in the cell culture supernatant and in the serum (FBS), bovine calf serum, penicillin and streptomycin were pur- cell layer after 13 days of culture, as this period of time corresponds to chased from Lonza (Levallois-Perret, France). β-Aminopropionitrile the highest expression of lysyl oxidase [23] using a fluorometric assay (BAPN) was purchased from VWR (Fontenay-sous-bois, France). All developed by Palamakumbura et al. [28]. At day 12, the differentiation other chemicals were purchased from Sigma-Aldrich (Lyon, France) medium was replaced by phenol red-free DMEM containing 0.5% when otherwise stated. bovine serum albumin, 50 μg/mL ascorbic acid, 2.5 mM β-glycerophos- phate and various concentrations of GIP. After 24 h, cell culture super- natants were collected, centrifuged at 3000 rpm for 30 min at 4 °C, Peptide synthesis aliquoted and stored at −80 °C until use. The cell layer was extracted in urea buffer as reported in [29]. Then, supernatants were incubated at 37 D-ala2-glucose-dependent insulinotropic polypeptide (GIP) was syn- °Cfor30mininthepresenceof1.2Murea,50mMsodiumborate thesized in our peptide supplier Genecust (Dudelange, Luxembourg). (pH 8.2), 1.3 nmol H2O2, 1 UI/mL horseradish peroxidase, 10 mM Briefly, GIP was sequentially synthesized using standard solid-phase diaminopentane, 10 μMAmplifluTM red and ± 0.5 mM BAPN in opaque Fmoc protocols [24] using Wang resin. Substitution of alanine at the 96 well plates. At the end of the incubation period, the plate was placed N-terminal position 2 with its (D) isomer was performed to ensure bet- on ice and fluorescence was read using a M2 microplate reader (Molecular ter resistance to serum dipeptidylpeptidase-4 as described elsewhere devices, St Gregoire, France) with excitation and emission wavelength set [25]. The peptide was then purified by reverse-phase HPLC (purity up at 563 nm and 587 nm, respectively. As BAPN is a specific lysyl oxidase N95%) using a SinoChrom ODS-BP column (4.6 × 250 mm) and subse- inhibitor, the residual activity evidenced in the quently characterized by electrospray ionization mass spectroscopy presence of BAPN was subtracted from the activity observed in the (ESI-MS) using a LCMS-2010 device (Shimadzu Corporation, Duisburg, absence of BAPN. Lysyl oxidase activity was reported as the fold Germany). Mass spectra were collected using full ion scan mode over change versus CTRL of the pooled supernatant and cell layer the mass-to-charge (m/z) range of 500–1800. Observed molecular fractions. mass for GIP was 4982.6 Da corresponding to theoretical value of 4983.6 Da [26]. GIP was then dissolved in phosphate buffer saline at a Western blotting concentration of 10−5 M, aliquoted and stored at −80 °C until use. MC3T3-E1cellswerewerewashedincoldPBSandlysatesweremade by using a lysis buffer containing 50 mM Tris–HCl pH 7.5, 100 mM NaCl,

Cell culture 50 mM NaF, 3 mM Na3VO4, protease inhibitor cocktail and 1% Nonidet P-40. Samples were spun at 13,000 rpm for 30 min at 4 °C, the superna- MC3T3-E1 cells are phenotypically normal osteoblasts derived from tant was collected and concentration was determined by BCA fetal mouse calvaria and differentiate in culture resulting ultimately assay (ThermoScientific, Brebières, France). Samples (20 μg per lane) to the formation of a bone-like [27]. MC3T3-E1 were run on a 10% acrylamide gel and blotted onto a PVDF membrane. cells were purchased from American type culture collection (ATCC, The membranes were washed in Tris buffered saline (TBS) and blocked Teddington, UK). Cells were grown and expanded at a ratio 1:4 in prop- with 5% bovine serum albumin. Samples were incubated overnight agation medium containing αMEM supplemented with 5% FBS, 5% bo- with the anti-GIPR (reference sc-98795, Santa Cruz Biotechnology, vine calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a Heidelberg, Germany) or β-actin (Sigma-Aldrich). Subsequently mem- humidified atmosphere enriched with 5% CO2 at 37 °C. For differentia- branes were washed in TBS and incubated with the appropriate second- tion studies, cells were detached with trypsin-EDTA and plated at a den- ary antibodies coupled to HRP (R&D Systems Europe). Immunoreactive sity of 1.5 × 104 cells/cm2 and grown to confluence in propagation bands were visualized using an ECL kit (Amersham, UK). medium. At confluence, the propagation medium was replaced by the differentiation medium containing αMEM supplemented with 5% FBS, Fourier transformed infrared microscopy (FTIRM) 5% bovine calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL ascorbic acid, 2.5 mM β glycerophosphate and various For collagen cross-link analysis, the same osteoblast cultures used for concentrations of GIP. This day was considered as day 1. 2′,5′- LOX activity were processed. Osteoblast cultures were fixed in absolute

Dideoxyadenosine (DDA) was used as an adenylyl cyclase inhibitor at ethanol, scrapped off the culture dish and transferred onto BaF2 windows a concentration of 50 μM. This concentration has been chosen based where they were air-dried. The integrity of the collagen extracellular ma- on preliminary results showing 95% reduction in adenylyl cyclase activ- trix was verified by comparing the obtained FTIRM spectrum with those ity and cAMP production without affecting osteoblast viability. The dif- of commercial collagen. Spectral analysis was performed using a Bruker ferentiation medium was replenished every two days. Vertex 70 spectrometer (Bruker optics, Ettlingen, Germany) interfaced A. Mieczkowska et al. / Bone 74 (2015) 29–36 31 with a Bruker Hyperion 3000 infrared microscope equipped with a stan- was computed and only fibrils with an AR b 1.2 were considered with dard single element Mercury Cadmium Telluride (MCT) detector. Infra- low probability of local tilting. The minor diameter of fitted ellipse red spectra were recorded at a resolution of 4 cm−1, with an average was selected as the value of fibril diameter as described by Starborg of 32 scans in transmission mode. Background spectral images were col- et al. [35].Morethan800fibrils were measured per condition. The di- lected under identical conditions from the same BaF2 windows at the be- ameter distribution was then analyzed with a lab-made routine using ginning and end of each experiment to ensure instrument stability. For ImageJ 1.45s and Excel 2010. Rapidly, the frequency of occurrence of FTIRM analysis, 20 spectra were acquired for each condition and ana- an i diameter was calculated as follows: lyzed with the Opus Software (release 6.0, Bruker). Water vapor was corrected prior to baseline correction. Spectra were then subjected to ¼ Ni Fi 100 second derivative spectroscopy and curve fitting routines using a com- Nt mercial available software package (Grams/AI 8.0, Thermofisher scientif- ic, Villebon sur Yvette, France) as described previously [30].Thecollagen where Ni represents the number of fibrils with the i diameter and Nt maturity index was determined as the relative ratio of pyridinium triva- represents the total number of fibrils. The frequency distribution, as a lent (Pyr, mature collagen) to dehydrodihydroxylysinonorleucine function of fibril diameter, was plotted and the following parameters divalent (deH-DHLNL, new collagen) collagen cross-links using their re- were deduced from this distribution: Dpeak corresponding to the most −1 −1 spective subbands located at 1660 cm and 1690 cm of the amide I frequently observed fibril diameter, Dmean, the average fibril diameter peak [31,32]. The -to-matrix ratio was calculated as the and Dwidth the width of the histogram at half maximum of the peak. −1 ratio of the 1376 cm peak, corresponding to the CH3 symmetric bend- ing vibration of GAGs [33], to the Amide I band (1585–1725 cm−1). The Statistical analysis sulfated proteoglycan-to-matrix ratio was calculated as the ratio of the −1 − 1064 cm peak, corresponding to SO3 symmetric stretching vibration Each experiment has been replicated at least 3 times. Results were of sulfated proteoglycan [34] to the Amide I band. expressed as mean ± standard error of the mean (SEM). Non- parametric Mann–Whitney U-test was used to compare the differences Transmission electron microscopy (TEM) between the groups using the Systat statistical software release 13.0 (Systat software Inc., San Jose, CA). Differences at p b 0.05 were consid- After 13 days of culture, osteoblasts and their produced extracellular ered to be significant. matrix were rinsed with 0.1 M cacodylate buffer (pH 7.4) and fixed in 2.5% glutaraldehyde in cacodylate buffer. Then cultures were post- Results fixed with 1% osmium tetroxide/1% potassium ferrocyanide and dehydrated in a graded series of ethanol prior to embedding in epoxy GIP affects lysyl oxidase (LOX) activity and collagen maturity resin. Ultrathin sections were cut with a diamond knife, contrasted with 3% uranyl acetate and observed with a Jeol JEM 1400 (Jeol, Croisy As represented Fig. 1A, GIPR is expressed in MC3T3-E1 osteoblast sur Seine, France) operating with an accelerating voltage of 120 KeV cells. GIP dose dependently induced a significant cAMP stimulation to and a magnification of ×30,000 (field overview) or ×100,000 (collagen reach 4 pmoles/well at concentrations of 1 nM and above and an EC50 network). of 96pM (Fig. 1B). We next investigated LOX activity in the presence of GIP ranged between 10 and 100 pM (Fig. 1C). LOX activity was aug- Characterization of collagen fibril diameter mented in cultures treated with as low as 10 pM GIP (1.1-fold increase, p b 0.05) as compared with untreated cells. Furthermore, LOX activity fi b b In order to avoid confounding factors due to angle incidence during was signi cantly increased by 2.2-fold (p 0.001), 3.6-fold (p 0.001) b sectioning, image analysis with shape descriptors was used to deter- and 3.9-fold (p 0.001) in the presence of 20 pM, 50 pM and 100 pM mine the diameter of cross-sectioned collagen fibrils. TEM photographs GIP, respectively. were subjected to image analysis using ImageJ 1.45s. sections We then examined the extent of mature and immature collagen were fitted with ellipses to evidence the presence of local tilting. The as- cross-links in the extracellular matrices (Fig. 2). The area of the −1 pect ratio (AR), defined by the following equation, 1690 cm subband, representative of the deH-DHLNL immature colla- gen cross-link, was dose-dependently augmented in GIP-treated culture (Fig. 2A). A similar pattern was observed with the area of the Diameter major axis 1660 cm−1, a peak representative of pyridinoline collagen cross-links. AR ¼ diameter minor axis The augmentation of the 1660 cm−1 area was even more marked

(A) (B) (C)

Fig. 1. GIP affects LOX activity and collagen maturity. (A) GIPR expression has been assessed by western blotting in three independent MC3T3 cell culture. (B) cAMP production in response to increasing doses of GIP. (C) LOX activity is dose-dependently increased in the presence of GIP. *: p b 0.05 and ***: p b 0.001 vs. untreated cultures. 32 A. Mieczkowska et al. / Bone 74 (2015) 29–36 (A) (B) (C)

(D) (E)

Fig. 2. GIP affects collagen maturity. (A) Amount of deH-DHLNL, (B) amount of Pyr and (C) collagen maturity index, expressed as the ratio of Pyr/deH-DHLNL (1660 cm−1/1690 cm−1), are dose-dependently increased in the presence of GIP. (D) Total proteoglycan content and (E) sulfated proteoglycan content in the extracellular bone matrix. *: p b 0.05, **: p b 0.01 and ***: p b 0.001 vs. untreated cultures.

at higher dose of GIP (Fig. 2B). The collagen maturity index was not sig- presence of 100 pM GIP (Fig. 3B). Indeed, Dmean, the mean diameter, nificantly different between vehicle- and 10 pM GIP-treated cultures and Dpeak, the most often observed diameter, were dramatically re- (p = 0.075) (Fig. 2C). But, at higher concentrations of GIP, significant duced, in a dose-dependent manner, in the presence of GIP with lower augmentations in this index were observed with 73% (p = 0.047), diameters observed at the highest tested concentrations (Table 1). On

127% (p = 0.009) and 131% (p = 0.009) increases in this parameter the other hand, Dwidth that characterizes heterogeneity in fibril diame- with 20 pM, 50 pM and 100 pM, respectively. We also assessed whether ters was not significantly different between untreated and GIP-treated GIP treatment affected the proteoglycan content of the extracellular ma- cultures at all tested concentrations (Table 1). trix. The PG-to-matrix and sulfated PG-to-matrix ratios were unaffected by the presence of GIP (Figs. 2DandE). Adenylyl cyclase activity is required for GIP-mediated increases in LOX activity and collagen crosslinking GIP affects collagen fibril diameters As binding of GIP to its receptor in osteoblasts leads to activation of As represented in Fig. 3A, it seemed that fibril diameters were re- adenylyl cyclase and production of cAMP, we ascertained whether the duced in the presence of 100 pM GIP. The frequency distribution of fibril observed increases in LOX activity and collagen cross-linking required diameters also appeared shifted towards lower diameter in the a functional adenylyl cyclase. The use of the adenylyl cyclase inhibitor

(A) (B)

Fig. 3. GIP affects collagen fibril diameter. (A) TEM photographs of untreated (left) and 100 pM GIP-treated (right) cells. Bar = 200 nm. Inset represents a higher magnification of collagen fibrils. Bar in inset = 100 nm. (B) Distribution frequency of fibril diameter between untreated (black) and 100 pM GIP-treated (gray) cells. Note the shift toward lower values for fibril diameter in GIP-treated cells. A. Mieczkowska et al. / Bone 74 (2015) 29–36 33

Table 1 Adenylyl cyclase activity is required for GIP-mediated modifications of the Properties of collagen fibrils in the presence of GIP. collagen matrix

GIP (pM) Dpeak (nm) Dmean (nm) Dwidth (nm) As represented Fig. 5,thefibril diameter seemed unmodified in con- – 32.7 ± 0.3 33.3 ± 0.5 10.7 ± 0.8 fi 10 30.3 ± 0.7* 29.7 ± 0.5** 12.3 ± 2.6 trol cultures treated with or without DDA. On the other hand, the bril di- 20 28.5 ± 0.3** 29.3 ± 0.8** 10.4 ± 1.2 ameter in GIP-treated cultures seemed higher in the presence of DDA. The 50 27.9 ± 0.8** 27.9 ± 0.2** 9.6 ± 0.5 frequency distribution of fibril diameter was unaffected in control 100 27.8 ± 0.7** 27.8 ± 0.5** 9.5 ± 0.7 cultures by the presence of DDA whilst the frequency distribution in *: p b 0.05 and **: p b 0.01 vs. cells cultured in the absence of GIP. GIP-treated cultures in the presence of DDA was broadened and shifted toward higher values. Not surprisingly, GIP treatment led to a decrease

in Dpeak and Dmean as reported above whilst Dwidth was unmodified Table 2 (Table 2). In control cultures, the addition of DDA did not affect any of Properties of collagen fibrils in the presence of an adenylyl cyclase inhibitor. Osteoblast the distribution frequency parameters. However, addition of DDA in cultures were treated with vehicle or 100 pM GIP in the presence of 50 μMDDA. GIP-treated cultures resulted to significant increases in Dmean and Dwidth Treatment Dpeak (nm) Dmean (nm) Dwidth (nm) but not Dpeak. The addition of DDA in GIP-treated cultures led to values fi Vehicle 32.0 ± 0.7 32.7 ± 0.6 11.8 ± 1.3 for Dpeak and Dwidth not signi cantly different from those observed in Vehicle + DDA 32.0 ± 1.2 32.7 ± 1.1 12.5 ± 0.8 control cultures. GIP 28.0 ± 0.2# 27.7 ± 0.2# 10.4 ± 0.8 # GIP + DDA 29.1 ± 0.8 30.2 ± 0.2** 13.8 ± 0.6 * Discussion *: p b 0.05 and **: p b 0.01 vs. cells cultured in the absence of DDA. # p b 0.05 vs. vehicle. Previously, in animal models of GIPR deficiency or stable GIP analog treatment, evidences have been provided that maturity of the collagen bone matrix was modified by the GIP/GIPR pathway [11,12]. However 2′,5′-dideoxyadenosine (DDA) significantly decreased the production of due to the pleiotropic expression of GIPR, it was unclear whether this cAMP in GIP-treated cultures (Fig. 4A). Furthermore, DDA had no influ- modification of collagen maturity resulted from direct action of the ence on LOX activity in vehicle treated-cultures (p = 0.507, Fig. 4B). On GIP/GIPR pathway on bone cells. In the present study, using a validated the other hand, this activity was reduced by 25% in the presence of DDA model of osteoblast cultures, we evidenced a significant stimulation of in GIP-treated cultures (p b 0.01, Fig. 4B). As a consequence, addition of cAMP production in response to increasing doses of GIP, significant aug- DDA in 100 pM GIP-treated cultures resulted in reductions in both mentations in LOX activity and collagen maturity in GIP-treated cultures − − 1690 cm 1 and 1660 cm 1 area, suggesting lower amount of immature and significant reductions in collagen fiber diameters. These modifica- and mature collagen cross-link, respectively (Figs. 4C and D). The colla- tions of LOX activity and collagen properties were partly due to stimula- gen maturity index was also significantly reduced in the presence of tion of adenylyl cyclase and production of cAMP as evidenced by DDA and 100 pM GIP (−36%, p = 0.016, Fig. 4E) as compared with alterations of these modifications in the presence of the adenylyl cyclase GIP alone-treated cultures. inhibitor, 2′,5′-dideoxyadenosine.

(A) (B)

(C) (D) (E)

Fig. 4. GIP effects on LOX activity and collagen maturity are due to a cAMP-dependent mechanism. (A) cAMP stimulation in 100 pM GIP culture is abolished by the use of 50 μM2′,5′- dideoxyadenosine (DDA). (B) Increase in LOX activity observed in the presence of 100 pM GIP is significantly reduced in DDA-treated cultures. White bars = untreated or GIP-treated cultures in the absence of DDA, black bars = untreated or GIP-treated cultures in the presence of DDA. (C) Amount of deH-DHLNL, (D) amount of Pyr and (E) collagen maturity index are significantly reduced in 100 pM GIP-treated cultures in the presence of DDA. White bars = untreated or GIP-treated cultures in the absence of DDA, black bars = untreated or GIP- treated cultures in the presence of DDA. *: p b 0.05 vs. cells cultured in the absence of DDA, **: p b 0.01 and ***: p b 0.001 vs. cells cultured in the absence of DDA. 34 A. Mieczkowska et al. / Bone 74 (2015) 29–36

(A) (B)

(C)

Fig. 5. GIP effects on collagen fibril diameter are due to a cAMP-dependent mechanism. (A) TEM photographs of extracellular matrices in vehicle, vehicle + DDA, GIP and GIP + DDA con- ditions. Bar = 100 nm. (B) Frequency distribution of fibril diameter in vehicle (black) and vehicle + DDA (gray) cultures. (C) Frequency distribution of fibril diameter in GIP (black) and GIP + DDA (gray) cultures.

Collagen must be cross-linked to exhibit the normal physical proper- Not only collagen maturation but also collagen fibril diameter was ties of tensile strength. The final enzymatic step of collagen cross-linking affected by GIP treatment. Indeed, we observed a significant 17% reduc- is ensured by LOX, an with amine oxidase activity, generating tion in the mean fibril diameter in the presence of 100 pM GIP. However, peptidyl-δ-hydroxy-δ-aminoadipic-δ-semialdehyde and peptidyl-α- the same heterogeneity in fibril diameter distribution was observed be- aminoadipic-δ-semialdehyde from hydroxylysine and residues, tween control and GIP-treated cultures as determined by the Dwidth pa- respectively [36]. These aldehydes then spontaneously react with rameter. Collagen fibril assembly was thought to be an entropy-driven other aldehydes or unmodified lysine and hydroxylysine residues to process as observed for microtubules and actin filament. On the other form a variety of intra- and intermolecular cross-links in . hand, some non-collagenous proteins have been implicated in fibrils LOX is synthesized in osteoblast as a pro-enzyme and excreted in the ex- assembly. Among them, biglycan and decorin, two of tracellular environment as a pro-enzyme. The pro-enzyme is then proc- the extracellular matrix have been suggested. Biglycan and decorin essed by procollagen C-proteinases in the extracellular environment to are composed of a protein core with leucin-reach repeats attached to give rise to the active 32 kDa enzyme and an 18 kDa propeptide [37, chains of . To determine the content of total proteo- 38]. Additionally, four LOX-like (LOXL1-4) have been identified glycans in the extracellular matrix we chose a spectroscopic approach and present significant sequence identity with mature LOX [39–44].In and we used the 1376 cm−1 band instead of the conventional 940– MC3T3-E1 cells, LOX, LOXL1, LOXL3 and LOXL4 have been shown to be 1140 cm−1 region. Indeed, although the 940–1140 cm−1 area com- the prominent form at the RNA level [45].Furthermore,LOXL1,LOXL3 prises the C\OH and C\C ring vibrations of the carbohydrate in proteo- and LOXL4 also present amine oxidase activities [46–48]. In the present glycans, this region might also contain information from plastic particles study, the LOX activity was determined in conditioned media of MC3T3- detached during extracellular matrix scrapping and ethanol residues E1 cells stimulated with various concentrations of GIP. We observed a (mainly between 940 and 1050 cm−1). Recently, Rieppo et al. demon- significant increase in this parameter; however it is unclear whether strated that the ratio of the 1376 cm−1 band over the entire integrated this observed increased is related to increases in only LOX or LOXL activ- area of the Amide I peak represented a good indicator of total proteogly- ities. Furthermore, it is also unclear whether the inhibitory action of DDA can content [33]. Furthermore, these authors made also the demonstra- on LOX activity reflects direct inhibition of LOX or LOXLs activities or a tion of the usefulness of the 1064 cm−1 band as a marker of sulfated combination of both. However, it is worth noting that in lox −/− proteoglycans [33]. The overall proteoglycan content and the sulfated animals, significant reductions in LOXLs at the RNA levels were observed proteoglycan content of the extracellular matrix were determined and [49]. Nevertheless, the increase in LOX activity after GIP stimulation was appeared unaffected by GIP treatment. As such, it is unlikely, although accompanied by an augmentation in collagen cross-linking as deter- we did not determine the expression of biglycan and decorin in the ex- mined spectroscopically. The ratio between the subbands located at tracellular matrix, that the observed reductions in collagen fibril diame- 1660 cm−1 and 1690 cm−1 is a cheap and validated methodology for ters were due to a modified pattern of proteoglycan expression. the determination of collagen cross-linking [32]. This methodology re- The GIP receptor has been shown to be expressed and functional in quired second-derivative spectral deconvolution and curve fitting in several osteoblastic cell lines (Saos-2, TE-85, MG-63) and primary mu- order to resolve underlying bands of the amide I peak. However, it can rine osteoblasts [6,8]. The GIP receptor is also expressed on MC3T3-E1 be applied either on tissue sections or cell culture material as we per- cells and addition of GIP in the cultures resulted in a significant stimula- formed in the present study [50–56]. tion of cAMP with an EC50 around 100 pM. The observed EC50 is A. Mieczkowska et al. / Bone 74 (2015) 29–36 35 relatively close to the physiological circulating plasma levels of GIP en- [20] Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J – fi 1996;316(Pt 1):1 11. countered after a meal. The use of a speci c adenylyl cyclase inhibitor, [21] Corsi A, Xu T, Chen XD, Boyde A, Liang J, Mankani M, et al. Phenotypic effects of 2′,5′-dideoxyadenosine was capable of reducing by 88%, the production biglycan deficiency are linked to collagen fibril abnormalities, are synergized by of cAMP in MC3T3-E1 cells in response to GIP. Moreover, DDA did not decorin deficiency, and mimic Ehlers–Danlos-like changes in bone and other con- fi nective tissues. J Bone Miner Res 2002;17:1180–9. only abolish the cAMP response but also contributed to signi cant re- [22] Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV. Targeted ductions in LOX activity (−25%) and collagen cross-linking (−36%). disruption of decorin leads to abnormal collagen fibril morphology and fragility. Furthermore, collagen deposition was also affected by the use of DDA J Cell Biol 1997;136:729–43. in GIP-treated cultures and significant augmentations in the mean fibril [23] Hong HH, Pischon N, Santana RB, Palamakumbura AH, Chase HB, Gantz D, et al. A role for lysyl oxidase regulation in the control of normal collagen deposition in dif- diameter and distribution heterogeneity were evidenced. Taken togeth- ferentiating osteoblast cultures. J Cell Physiol 2004;200:53–62. er, these data support a direct role for a GIPR-adenylyl cyclase-cAMP [24] FieldsGB,NobleRL.Solidphasepeptidesynthesisutilizing9-fluorenylmethoxycarbonyl – axis in the control of extracellular matrix properties in response to amino acids. Int J Pept Protein Res 1990;35:161 214. [25] Kuhn-Wache K, Manhart S, Hoffmann T, Hinke SA, Gelling R, Pederson RA, et al. An- GIP. Noteworthy is the residual cAMP activity in the presence of GIP/ alogs of glucose-dependent insulinotropic polypeptide with increased dipeptidyl DDA. Indeed, in this condition, MC3T3-E1 exhibited still a 7% increase peptidase IV resistance. Adv Exp Med Biol 2000;477:187–95. in cAMP as compared with untreated cells. One could wonder why we [26] McIntosh CH, Widenmaier S, Kim SJ. Glucose-dependent insulinotropic polypeptide (Gastric Inhibitory Polypeptide; GIP). Vitam Horm 2009;80:409–71. did not use higher dose of DDA to completely abolish the rise in cAMP. [27] Franceschi RT, Iyer BS, Cui Y. Effects of ascorbic acid on collagen matrix formation As a preliminary experiment, we used several doses of DDA and and osteoblast differentiation in murine MC3T3-E1 cells. J Bone Miner Res 1994;9: 50 μM was the highest dose usable as higher concentration led to cell 843–54. [28] Palamakumbura AH, Trackman PC. A fluorometric assay for detection of lysyl oxi- death in the culture. Of course, we cannot rule out that other intracellu- dase enzyme activity in biological samples. Anal Biochem 2002;300:245–51. lar pathways, independent of adenylyl cyclase activation, might also be [29] Bedell-Hogan D, Trackman P, Abrams W, Rosenbloom J, Kagan H. Oxidation, cross- stimulated by the binding of GIP to the GIPR and as such might contrib- linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase. J Biol Chem 1993;268:10345–50. ute to the observed modifications of extracellular matrix. [30] Mabilleau G, Mieczkowska A, Irwin N, Flatt PR, Chappard D. Optimal bone mechan- In conclusion, the present study supports a direct role for the GIPR- ical and material properties require a functional GLP-1 receptor. J Endocrinol 2013; adenylyl cyclase-cAMP in the control of extracellular matrix deposition 219:59–68. and properties in osteoblasts. GIP-treated osteoblasts presented with [31] Paschalis EP, Gamsjaeger S, Tatakis DN, Hassler N, Robins SP, Klaushofer K. Fourier transform infrared spectroscopic characterization of mineralizing augmented LOX activity and collagen cross-linking as well as modifica- enzymatic trivalent cross-links. Calcif Tissue Int 2014. http://dx.doi.org/10.1007/ tion of collagen deposition. s00223-014-9933-9. [32] Paschalis EP, Verdelis K, Doty SB, Boskey AL, Mendelsohn R, Yamauchi M. Spectroscop- ic characterization of collagen cross-links in bone. J Bone Miner Res 2001;16:1821–8. [33] Rieppo L, Saarakkala S, Narhi T, Helminen HJ, Jurvelin JS, Rieppo J. Application of sec- References ond derivative spectroscopy for increasing molecular specificity of Fourier transform infrared spectroscopic imaging of articular cartilage. Osteoarthritis Cartilage 2012; [1] Andersen DK, Elahi D, Brown JC, Tobin JD, Andres R. Oral glucose augmentation of 20:451–9. insulin secretion. Interactions of gastric inhibitory polypeptide with ambient glucose [34] Servaty R, Schiller J, Binder H, Arnold K. Hydration of polymeric components of car- and insulin levels. J Clin Invest 1978;62:152–61. tilage—an infrared spectroscopic study on hyaluronic acid and chondroitin sulfate. [2] Schirra J, Katschinski M, Weidmann C, Schafer T, Wank U, Arnold R, et al. Gastric Int J Biol Macromol 2001;28:121–7. emptying and release of incretin hormones after glucose ingestion in humans. [35] Starborg T, Kalson NS, Lu Y, Mironov A, Cootes TF, Holmes DF, et al. Using transmis- J Clin Invest 1996;97:92–103. sion electron microscopy and 3View to determine collagen fibril size and three- [3] Varner AA, Isenberg JI, Elashoff JD, Lamers CB, Maxwell V, Shulkes AA. Effect of intra- dimensional organization. Nat Protoc 2013;8:1433–48. venous lipid on gastric acid secretion stimulated by intravenous amino acids. [36] Trackman PC. Diverse biological functions of extracellular collagen processing Gastroenterology 1980;79:873–6. enzymes. J Cell Biochem 2005;96:927–37. [4] Jorde R, Burhol PG, Schulz TB. Fasting and postprandial plasma GIP values in man [37] Panchenko MV, Stetler-Stevenson WG, Trubetskoy OV, Gacheru SN, Kagan HM. measured with seven different antisera. Regul Pept 1983;7:87–94. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl [5] Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; oxidase. Potential role of procollagen C-proteinase. J Biol Chem 1996;271:7113–9. 132:2131–57. [38] Uzel MI, Scott IC, Babakhanlou-Chase H, Palamakumbura AH, Pappano WN, Hong [6] Bollag RJ, Zhong Q, Phillips P, Min L, Zhong L, Cameron R, et al. Osteoblast-derived HH, et al. Multiple bone morphogenetic protein 1-related mammalian metallopro- cells express functional glucose-dependent insulinotropic peptide receptors. teinases process pro-lysyl oxidase at the correct physiological site and control Endocrinology 2000;141:1228–35. lysyl oxidase activation in mouse embryo fibroblast cultures. J Biol Chem 2001; [7] Mabilleau G, Gaudin-Audrain C, Irwin N, Flatt PR, Basle MF, Chappard D. Deficiency 276:22537–43. in glucose-dependent insulinotropic peptide receptor results in higher bone mass in [39] Huang Y, Dai J, Tang R, Zhao W, Zhou Z, Wang W, et al. Cloning and characterization male mice. Osteoporos Int 2012;23:S407–8. of a human lysyl oxidase-like 3 gene (hLOXL3). Matrix Biol 2001;20:153–7. [8] Pacheco-Pantoja EL, Ranganath LR, Gallagher JA, Wilson PJ, Fraser WD. Receptors [40] Ito H, Akiyama H, Iguchi H, Iyama K, Miyamoto M, Ohsawa K, et al. Molecular cloning and effects of gut hormones in three osteoblastic cell lines. BMC Physiol 2011;11:12. and biological activity of a novel lysyl oxidase-related gene expressed in cartilage. [9] Zhong Q, Itokawa T, Sridhar S, Ding KH, Xie D, Kang B, et al. Effects of glucose- J Biol Chem 2001;276:24023–9. dependent insulinotropic peptide on osteoclast function. Am J Physiol Endocrinol [41] Jourdan-Le Saux C, Le Saux O, Donlon T, Boyd CD, Csiszar K. The human lysyl Metab 2007;292:E543–8. oxidase-related gene (LOXL2) maps between markers D8S280 and D8S278 on [10] Gaudin-Audrain C, Irwin N, Mansur S, Flatt PR, Thorens B, Basle M, et al. Glucose- 8p21.2–p21.3. Genomics 1998;51:305–7. dependent insulinotropic polypeptide receptor deficiency leads to modifications of [42] Kenyon K, Modi WS, Contente S, Friedman RM. A novel human cDNA with a predict- trabecular bone volume and quality in mice. Bone 2013;53:221–30. ed protein similar to lysyl oxidase maps to chromosome 15q24–q25. J Biol Chem [11] Mieczkowska A, Irwin N, Flatt PR, Chappard D, Mabilleau G. Glucose-dependent 1993;268:18435–7. insulinotropic polypeptide (GIP) receptor deletion leads to reduced bone strength [43] Maki JM, Kivirikko KI. Cloning and characterization of a fourth human lysyl oxidase and quality. Bone 2013;56:337–42. isoenzyme. Biochem J 2001;355:381–7. [12] Mabilleau G, Mieczkowska A, Irwin N, Simon Y, Audran M, Flatt PR, et al. Beneficial [44] Maki JM, Tikkanen H, Kivirikko KI. Cloning and characterization of a fifth human effects of a N-terminally modified GIP agonist on tissue-level bone material proper- lysyl oxidase isoenzyme: the third member of the lysyl oxidase-related subfamily ties. Bone 2014;63C:61–8. with four scavenger receptor cysteine-rich domains. Matrix Biol 2001;20:493–6. [13] Chappard D, Basle MF, Legrand E, Audran M. New laboratory tools in the assessment [45] Atsawasuwan P, Mochida Y, Parisuthiman D, Yamauchi M. Expression of lysyl of bone quality. Osteoporos Int 2011;22:2225–40. oxidase isoforms in MC3T3-E1 osteoblastic cells. Biochem Biophys Res Commun [14] Currey JD. The mechanical consequences of variation in the mineral content of bone. 2005;327:1042–6. JBiomech1969;2:1–11. [46] Kim MS, Kim SS, Jung ST, Park JY, Yoo HW, Ko J, et al. Expression and purifi cation of [15] Currey JD. The effect of porosity and mineral content on the Young's modulus of enzymatically active forms of the human lysyl oxidase-like protein 4. J Biol Chem elasticity of compact bone. J Biomech 1988;21:131–9. 2003;278:52071–4. [16] Wang X, Bank RA, TeKoppele JM, Agrawal CM. The role of collagen in determining [47] Kim S, Kim Y. Variations in LOXL1 associated with exfoliation glaucoma do not affect bone mechanical properties. J Orthop Res 2001;19:1021–6. amine oxidase activity. Mol Vis 2012;18:265–70. [17] Wang X, Shen X, Li X, Agrawal CM. Age-related changes in the collagen network and [48] Lee JE, Kim Y. A tissue-specific variant of the human lysyl oxidase-like protein 3 toughness of bone. Bone 2002;31:1–7. (LOXL3) functions as an amine oxidase with substrate specificity. J Biol Chem [18] Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for 2006;281:37282–90. therapy. Annu Rev Biochem 1995;64:403–34. [49] Pischon N, Maki JM, Weisshaupt P, Heng N, Palamakumbura AH, N'Guessan P, et al. [19] Yamauchi M, Sricholpech M. Lysine post-translational modifications of collagen. Lysyl oxidase (lox) gene deficiency affects osteoblastic phenotype. Calcif Tissue Int Essays Biochem 2012;52:113–33. 2009;85:119–26. 36 A. Mieczkowska et al. / Bone 74 (2015) 29–36

[50] Mourant JR, Yamada YR, Carpenter S, Dominique LR, Freyer JP. FTIR spectroscopy [54] Thaler R, Zwerina J, Rumpler M, Spitzer S, Gamsjaeger S, Paschalis EP, et al. Homo- demonstrates biochemical differences in mammalian cell cultures at different cysteine induces serum amyloid A3 in osteoblasts via unlocking RGD-motifs in growth stages. Biophys J 2003;85:1938–47. collagen. FASEB J 2013;27:446–63. [51] Paschalis EP, Mendelsohn R, Boskey AL. Infrared assessment of bone quality: [55] Turecek C, Fratzl-Zelman N, Rumpler M, Buchinger B, Spitzer S, Zoehrer R, et al. Col- a review. Clin Orthop Relat Res 2011;469:2170–8. lagen cross-linking influences osteoblastic differentiation. Calcif Tissue Int 2008;82: [52] Ramesh J, Salman A, Hammody Z, Cohen B, Gopas J, Grossman N, et al. FTIR micro- 392–400. scopic studies on normal and H-ras oncogene transfected cultured mouse fibro- [56] Varga F, Rumpler M, Zoehrer R, Turecek C, Spitzer S, Thaler R, et al. T3 affects expres- blasts. Eur Biophys J 2001;30:250–5. sion of collagen I and collagen cross-linking in bone cell cultures. Biochem Biophys [53] Thaler R, Spitzer S, Rumpler M, Fratzl-Zelman N, Klaushofer K, Paschalis EP, et al. Dif- Res Commun 2010;402:180–5. ferential effects of homocysteine and beta aminopropionitrile on preosteoblastic MC3T3-E1 cells. Bone 2010;46:703–9.