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Am J Physiol Cell Physiol 292: C2095-C2102, 2007. First published February 7, 2007; doi:10.1152/ajpcell.00613.2006
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Intracellular distribution of the lysyl oxidase propeptide in osteoblastic cells

Ying Guo,1,* Nicole Pischon,1,2,* Amitha H. Palamakumbura,1 and Philip C. Trackman1,3

1Division of Oral Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts; 2Department of Periodontology, Charité Universitätsmedizin, Berlin, Germany; and 3Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts

Submitted 8 December 2006 ; accepted in final form 6 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lysyl oxidase plays a critical role in the formation of the extracellular matrix, and its activity is required for the normal maturation and cross-linking of collagen and elastin. An 18-kDa lysyl oxidase propeptide (LOPP) is generated from 50-kDa prolysyl oxidase by extracellular proteolytic cleavage during the biosynthesis of active 30-kDa lysyl oxidase enzyme. The fate and the functions of the LOPP are largely unknown, although intact LOPP was previously observed in osteoblast cultures. We investigated the spatial localization of molecular forms of lysyl oxidase, including LOPP in proliferating and differentiating osteoblasts, by using confocal immunofluorescence microscopy and Western blots of cytoplasmic and nuclear extracts. In the present study, a stage-dependent intracellular distribution of LOPP in the osteoblastic cell was observed. In proliferating osteoblasts, LOPP epitopes were principally associated with the Golgi and endoplasmic reticulum, and mature lysyl oxidase epitopes were found principally in the nucleus and perinuclear region. In differentiating cells, LOPP and mature lysyl oxidase immunostaining showed clear colocalization with the microtubule network. The subcellular distribution of LOPP and its temporal and physical association with microtubules were confirmed by Western blot and far Western blot studies. We also report that N-glycosylated and nonglycosylated LOPP are present in MC3T3-E1 cell cultures. We conclude that LOPP has a stage-dependent intracellular distribution in osteoblastic cells. Future studies are needed to investigate whether the LOPP associations with microtubules or the osteoblast nucleus have functional effects for osteoblast differentiation and bone formation.

microtubules; confocal immunofluorescence microscopy; extracellular matrix; osteoblast differentiation

LYSYL OXIDASE CATALYZES the oxidative deamination of peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin. The resulting aldehydes react further leading to covalent cross-link formation (19, 34). Collagen cross-links are required for its normal physical properties of tensile strength, and deficient levels of lysyl oxidase-dependent cross-links in bone collagen are associated with osteoporosis and weak bones (1, 23, 30, 31).

Lysyl oxidase is synthesized as a 50-kDa proenzyme (39). Prolysyl oxidase is N-glycosylated in the endoplasmic reticulum and the Golgi complex and is then secreted into the extracellular environment, where it is processed into the 32-kDa mature lysyl oxidase enzyme and an 18-kDa propeptide (LOPP) by bone morphogenetic protein (BMP)-1 family members (6, 33, 39, 41). In addition to its traditional role as an extracellular enzyme required for cross-link formation, lysyl oxidase was also found to have tumor suppressor activity (5, 21). Recent data indicate that LOPP is largely responsible for phenotypic reversion of ras-transformed fibroblasts and breast cancer tumor cells and tumor suppression by lysyl oxidase (17, 32), whereas lysyl oxidase enzyme activity is associated with promoting tumor invasiveness (9, 22). A new understanding emerging from these observations is that there may be a balance in the biological activities between the released LOPP and active lysyl oxidase enzyme. Recently, our group (15) reported that LOPP immunostaining was observed in osteoblast cultures and that intact LOPP was detected in the whole cell lysate of differentiating MC3T3-E1 cells. In the present study, we further investigated intracellular localization of LOPP at different stages of osteoblast differentiation by using confocal immunofluorescence microscopy and Western blot analyses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. {alpha}-Minimum essential medium ({alpha}-MEM), penicillin-streptomycin solution, nonessential amino acids, trypsin-EDTA, and Pro-Bond resin were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). Goat anti-rabbit Alexa Fluor 568, goat anti-mouse Alexa Fluor 488, BODIPY FL C5-ceramide, and Topro-3 were obtained from Molecular Probes (Eugene, OR).

Cell culture. Murine osteoblast-like MC3T3-E1 cells were obtained from the American Type Culture Collection. Cells were cultured in {alpha}-MEM containing 10% FBS, 1% nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin. To initiate differentiation, at confluence, cells were fed with {alpha}-MEM, 10% FBS, 50 µg/ml ascorbate, 1% nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin for 2 days. Beginning on day 0 of differentiation, cells were cultured in this medium containing, in addition, 10 mM beta-glycerophosphate.

Immunofluorescence analyses. MC3T3-E1 cells were cultured on Lab-Tek glass four-chamber slides in complete medium or differentiation medium. Subconfluent or differentiating cells were then fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were made permeable in ice-cold 0.2% Triton X-100 and blocked in 1.5% serum of the same species as the second antibody was generated. After blocking, slides were incubated with primary antibody for 2 h. Primary antibody was diluted in blocking serum. Nonimmune antisera or purified immunoglobulins were used as negative controls. Subsequently, slides were incubated with appropriate secondary antibody for 1 h. Cells were then double stained with different organelle markers (Alexa Fluor 488 concanavalin A for endoplasmic reticulum, BODIPY FL C5-ceramide for Golgi complex, and Topro-3 for nuclear staining) or phalloidin, for actin. Slides were mounted with Gel/Mount anti-fade aqueous mounting material (Biomeda) and observed with a Zeiss inverted LSM 510 confocal laser scanning microscope. Cell images were captured, and image analysis was performed using Zeiss LSM 510 software 2.5. Primary LOPP antibody was generated against recombinant LOPP expressed in Escherichia coli as previously described (15), and the IgY fraction was used at a concentration of 79 µg/ml. Mature anti-bovine lysyl oxidase antiserum was provided by the laboratory of Herbert M. Kagan (Boston University School of Medicine). Mouse anti-bovine tubulin monoclonal antibody (catalog no. A-11126; Molecular Probes) was used at a concentration of 1 µg/ml. Nonimmune controls gave no significant signals in the confocal analyses.

Cytosol and nuclear fraction preparation. Proliferating MC3T3-E1 cells from two confluent 100-mm cell culture plates were washed twice with ice-cold PBS, scraped into PBS, pelleted, and resuspended in 500 µl of Nonidet P-40 (NP-40) lysis buffer (0.5% IGEPAL CA 630, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-Cl, pH 7.6, and 1 mM PMSF). After incubation at 4°C for 20 min, the sample was centrifuged at 3,000 rpm at 4°C for 5 min. The resulting supernatant was the cytosolic fraction. The pellet was washed with cell lysis buffer three times and centrifuged at 3,000 rpm at 4°C for 5 min. The resulting pellet was dissolved in 50 µl of sample buffer and termed the nuclear fraction (3). For differentiating cells, two 100-mm plates of cells were washed with PBS and collected, and the suspension containing cells and extracellular matrix was centrifuged and suspended in lysis buffer, as described for proliferating cells. The pellet was suspended in 1 ml of lysis buffer and then gently homogenized with a Dounce homogenizer for 40 strokes on ice, incubated for 20 min, and processed as described for proliferating cells. All subsequent steps were identical to those described for proliferating cells. The cytosolic and nuclear fractions were subjected to immunoblotting. Protein concentrations were determined using the NanoOrange kit (Molecular Probes).

Immunoblotting. Cell fractions were resolved by electrophoresis on 10% tricine-SDS-PAGE gels. The proteins were then transferred to polyvinylidene difluoride membranes (38). Blots were blocked in TBST (10 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 0.5% Tween) containing 5% nonfat dry milk for 1 h and incubated with primary antibody diluted in TBST containing 5% nonfat dry milk overnight at 4°C. After extensive washing with TBST, the blots were incubated with secondary antibody for 1 h. Polyclonal anti-lysyl oxidase propeptide IgY was generated against recombinant propeptide fragment 23–146 (15). Affinity purified anti-mature lysyl oxidase antibody was generated against rat peptide sequences 181 to 196 and 293 to 309, as described previously (17). Appropriate secondary horseradish peroxidase-coupled secondary antibodies were utilized, obtained from Promega. Enhanced chemiluminescence (ECL; Amersham Pharmacia) was used to visualize the specific protein bands.

Preparation of recombinant proteins. Rat LOPP was expressed in E. coli as a fusion protein containing an NH2-terminal Xpress tag and was purified as described previously (15).

Deglycosylation assay. MC3T3-E1 cells were cultured in the presence or absence of 400 pM transforming growth factor (TGF)-beta for 18 h in 100-mm cell culture plates (10). Media samples were collected from five plates per group (50 ml) and concentrated to 0.5 ml with Centricon-plus (Amicon) concentrators, and then 100-µl aliquots were precipitated with cold acetone overnight at –20°C and pellets were collected by centrifugation in a refrigerated microcentrifuge at maximum speed for 20 min (14). Supernatant fluids were discarded, and the pellets were air dried. The pellets were redissolved in 90 µl of reaction buffer (20 mM sodium phosphate, pH 7.5) followed by 5 µl of 2% SDS and 1 M 2-mercaptoethanol to a final concentration of 0.1% SDS and 50 mM 2-mercaptoethanol. The reactions were boiled for 5 min and cooled to room temperature, and then 5 µl of nonionic detergent IGEPAL CA 630 were added, and 24-µl aliquots were treated with the N-glycosidase PNGase F at 37°C for 2 days, according to the product instructions (Sigma). Control reactions were treated with enzyme vehicle (20 mM Tris, pH 7.5, 1 mM EDTA, and 50 mM NaCl). After digestion, the samples were boiled in sample buffer and electrophoresed on 10% tricine-SDS-PAGE, followed by Western blot analysis with the anti-LOPP antibody.

Far Western blots. Tubulin overlay assays (far Western blots) to investigate tubulin-LOPP interactions were conducted using a procedure modified from Rozdzial et al. (35). Recombinant LOPP (10 µg) and bovine serum albumin (BSA, 10 µg; Sigma) were separated on SDS-PAGE and electrotransferred to nitrocellulose membranes. Blots were blocked in PBS containing 2% BSA for 2 h at room temperature. The blots were then incubated with 10 µg/ml tubulin (cytoskeleton) or polymerized tubulin in overlay buffer (1% BSA, 20% sucrose, 100 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.6–6.8, 1 mM DTT, 25 mM NaF, and protease inhibitor cocktail) at 4°C overnight. Tubulin polymerization was enhanced by incubating tubulin in the presence of 20 µM taxol and 1 mM GTP, incubated at 37°C for 30 min. The blots were then washed with PEMG buffer (100 mM PIPES, 1 mM EGTA, and 1 mM MgCl2) and fixed in 0.5% formaldehyde in PBS for 1 h. The reaction was stopped by incubation in 2% glycine in PBS for 30 min. The blots were washed in PBS, incubated with anti-tubulin antibody (Molecular Probes), and subjected to ECL detection as for Western blots.

LOPP pull-down assay. MC3T3-E1 cells were harvested on day 6 of differentiation. Cells were suspended in cell lysis buffer (20 mM Tris-Cl, pH 8.0, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% NP-40, 1 mM vanadate, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin) and homogenized on ice for 1 min at full speed (20). The lysate was clarified by centrifugation at maximum speed for 15 min at 4°C. The supernatant was then precleared by incubation with blank nickel affinity column beads overnight at 4°C (HIS-Select; Sigma) The cleared cell lysate was incubated with recombinant His-tagged LOPP immobilized on HIS-Select resin or with HIS-SELECT resin without bound LOPP for 2–3 h with continuous rocking at 4°C. Before the incubation, the beads were blocked in cell lysis buffer containing 3% BSA overnight at 4°C. Beads were washed six times with lysis buffer containing 20 mM imidazole. The bound proteins were then eluted with 50 µl of 0.1 M sodium phosphate, pH 4.3, and 8 M urea and subjected to Western blot analysis for tubulin.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of LOPP in proliferating MC3T3-E1 cells by immunofluorescence. Osteoblast differentiation exhibits a series of well-characterized phenotypic phases: proliferation, matrix accumulation, and mineralization (36). We first investigated the localization of LOPP epitopes in proliferating osteoblastic MC3T3-E1 cells by utilizing the antibody raised against the lysyl oxidase propeptide. Immunofluorescence studies performed with a confocal microscope revealed LOPP immunoreactivity primarily in the perinuclear region (Fig. 1A). To further characterize the distribution of LOPP, we performed multiple labeling using specific fluorochromes for cell organelles and cytoskeletal proteins. Immunofluorescence analyses showed that the perinuclear staining of LOPP was mostly in the Golgi complex (Fig. 1B), with more limited colocalization with the endoplasmic reticulum and no colocalization with actin or tubulin at this stage of differentiation (data not shown).


Figure 1
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  		Fig. 1. Immunofluorescence microscopy stained with antibodies against lysyl oxidase propeptide (LOPP) (A and B) and mature 30-kDa lysyl oxidase (C) in proliferating MC3T3-E1 cells shows different locations of molecular forms of lysyl oxidase. MC3T3-E1 cells were cultured, and exponentially growing cells were fixed, made permeable, and stained as described in MATERIALS AND METHODS. Cells in A and B were stained for LOPP with anti-LOPP IgY, followed by goat anti-chicken Alexa Fluor 568. In B, BODIPY FL C5-ceramide (green) was used to stain Golgi complex. In C, mature rabbit anti-lysyl oxidase antibody was visualized with goat anti-rabbit Alexa Fluor 568, and nuclei were stained with Topro-3 (blue).

 
Since LOPP antibody recognizes both the LOPP and lysyl oxidase proenzyme, we stained cells with a polyclonal antibody raised against mature ~30-kDa lysyl oxidase to gain insight into the localization of different molecular forms of lysyl oxidase. The immunoreactivity with the anti-mature lysyl oxidase antibody showed a different distribution from that of the LOPP. Intense staining occurred in the nucleus with the lysyl oxidase antibody, which was clearly demonstrated in merged images with the nuclear marker (Fig. 1C). Further analyses revealed weak staining with anti-mature lysyl oxidase antibody in the endoplasmic reticulum and in the Golgi complex, and analyses done with both the LOPP and anti-mature lysyl oxidase antibody did not reveal any colocalization with actin or tubulin at this stage of differentiation (data not shown).

Western blot detection of molecular forms of lysyl oxidase in cytoplasmic and nuclear fractions of MC3T3-E1 cells. To independently investigate the subcellular distribution of 50-kDa prolysyl oxidase, 32-kDa mature lysyl oxidase, and the lysyl oxidase propeptide, we next performed Western blot analyses of nuclear and cytoplasmic fractions of MC3T3-E1 cells. The purpose of these Western blots is to identify molecular forms of lysyl oxidase present in each fraction and is not quantitative. Thus, because nuclear extracts are made from nuclei recovered as pellets after centrifugation and are more concentrated than cytoplasmic supernatants, signals on Western blots from nuclear and cytoplasmic samples are not necessarily in proportion to the concentration of epitopes seen in whole cells with the use of immunofluorescence. In fractions made from proliferating MC3T3-E1 cells, Fig. 2A shows that the LOPP antibody detects three molecular forms of LOPP: the 50-kDa lysyl oxidase proenzyme and the 35- and 18-kDa forms of LOPP. The 50-kDa lysyl oxidase proenzyme was detected in both the cytoplasmic and nuclear fractions. Both the 35- and 18-kDa LOPP were detected in the nuclear fraction, whereas only the 35-kDa LOPP was detected in the cytosolic fraction.


Figure 2
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  		Fig. 2. Western blot analyses of molecular forms of lysyl oxidase cytosol and nuclear preparations made from proliferating MC3T3-E1 osteoblasts. In A, fractions were stained with anti-LOPP, and in B, with anti-mature lysyl oxidase. Purity of nuclear and cytoplasmic fractions was confirmed by analyses with anti-histone H1 (C) and anti-tubulin antibodies (D), respectively. The same samples were analyzed in A–D.

 
Analyses with the anti-mature 32-kDa lysyl oxidase antibody (Fig. 2B) detected mainly the 50-kDa proenzyme and smaller amounts of the 32-kDa mature enzyme in the nuclear fraction. Very low levels of 50-kDa proenzyme band or 32-kDa bands were detected in the cytosolic fraction with the lysyl oxidase antibody. This is because of the low amounts of lysyl oxidase proteins in the cytosol and is in agreement with immunofluorescence data (Fig. 1C) showing primarily nuclear localization of lysyl oxidase at this stage of differentiation. The identity of the weak ~40-kDa band in the cytosol fraction was not determined, but its weak intensity is consistent with low amounts of immunofluorescence seen in the cytosol with this antibody. The purity of the cytoplasmic and nuclear fractions, respectively, were analyzed with tubulin antibody as a cytosolic marker and histone H1 antibody as a nuclear marker. As expected, tubulin was only detected in the cytosolic fraction, whereas histone H1 was only detected in the nuclear fraction.

Localization of LOPP in differentiating MC3T3-E1 cells by immunofluorescence. To follow the distribution of LOPP during osteoblast development, we next investigated the localization of LOPP epitopes in differentiating MC3T3-E1 cells. Immunofluorescence analyses with the anti-LOPP antibody revealed that on day 6 of differentiation, LOPP immunoreactivity was mainly in the cytosol. Staining was also observed in nucleus, but this was lower in proportion compared with the degree of staining observed in the cytosol (Fig. 3A). Double immunofluorescence staining showed that cytosolic LOPP was largely colocalized with tubulin (Fig. 3A), suggesting that LOPP is associated with microtubules at this stage of differentiation. Cytosolic LOPP staining did not show significant colocalization with the ER and Golgi (data not shown), in contrast to what was seen in proliferating cells (Fig. 1). Thus there is a differentiation-stage dependence of the distribution of LOPP epitopes in developing osteoblasts in which LOPP epitopes are found mainly in the endoplasmic reticulum and Golgi in proliferating cells (Fig. 1) and in microtubules in differentiating cells (Fig. 3).


Figure 3
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  		Fig. 3. Immunofluorescence microscopy stained with antibodies against LOPP (A) and lysyl oxidase (B) in differentiating MC3T3-E1 cells. In B, cells were treated either with DMSO vehicle (–) or with 10 µM nocodazole (+) for 1 h at 37°C to further investigate microtubule associations with microtubules. In A, cells were permeabilized and then double-stained with anti-LOPP IgY and anti-tubulin IgG, followed by goat anti-chicken Alexa Fluor 488 (green) and goat anti-mouse Alexa Fluor 568 (red), respectively. In B, permeabilized cells were double-stained with rabbit anti-lysyl oxidase IgG and anti-tubulin IgG, followed by goat anti-rabbit Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488, respectively.

 
The immunoreactivity seen with the anti-30-kDa lysyl oxidase antibody in these differentiating MC3T3-E1 cells also showed a different distribution from that in proliferating cells. Predominant lysyl oxidase staining was localized with microtubules (Fig. 3B). Weak staining was observed in the nucleus. The colocalization of lysyl oxidase epitopes with microtubules was still present following tubule disruption with nocodazole treatment (Fig. 3B).

Western blot analyses of differentiating MC3T3-E1 cells. In differentiating MC3T3-E1 cells, all three molecular forms of LOPP were detected in both cytosolic and nuclear fractions of the differentiating cells in agreement with the immunofluorescence findings showing microtubule and nuclear associations of LOPP (data not shown). Anti-32-kDa lysyl oxidase antibody detected the 50-kDa proenzyme and the 32-kDa mature enzyme in the cytosolic fraction, and a much weaker 50-kDa proenzyme band was detected in the nuclear fraction (Fig. 4A), consistent with immunofluorescence studies (Fig. 3).


Figure 4
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  		Fig. 4. Western blot analysis of molecular forms of lysyl oxidase in cytosol and nuclear preparations made from differentiating MC3T3-E1 osteoblasts (A) and Western blots with anti-LOPP antibody of PNGase F-treated media proteins from 18-h TGF-beta1-treated and untreated control MC3T3-E1 cells (B). Cytosolic and nuclear fractions of MC3T3-E1 cells were obtained from cells cultured in differentiation medium for 6 days as described in MATERIALS AND METHODS. Both fractions were separated in SDS-PAGE and electroblotted to a polyvinylidene difluoride membrane. The resulting blots were detected with anti-mature lysyl oxidase antibodies (A). Tubulin was used as a cytosolic marker, and histone H1 was used as a nuclear marker. In B, lane 1 is recombinant LOPP (rLOPP) expressed and purified from Escherichia coli (15), and its mobility matches that of LOPP from media of MC3T3-E1 cells after treatment with PNGase F (lanes 3 and 5).

 
In summary, in proliferating cells, LOPP epitopes were found primarily in the perinuclear region, where they were associated with the Golgi and endoplasmic reticulum, whereas the lysyl oxidase antibody primarily marked the nucleus. In differentiating cells, LOPP and lysyl oxidase immunoreactivity both exhibited primarily microtubule associations. Thus the data show that as a function of differentiation, the major sites of accumulation of LOPP and lysyl oxidase epitopes, respectively, change.

The 35-kDa immunoreactive protein is N-glycosylated LOPP. As shown above, three specific immunoreactive bands were detected by anti-lysyl oxidase propeptide antibody in MC3T3-E1 cellular fractions. The 50-kDa protein corresponds to lysyl oxidase proenzyme; the 18-kDa mass matches the mobility of bacterial recombinant LOPP, although the calculated molecular weight of LOPP is 15,290. The structure of the 35-kDa immunoreactive protein is not immediately obvious, and this was next investigated. Our group (39) has previously shown that the 50-kDa proenzyme is N-glycosylated and that the 32-kDa mature enzyme was not, consistent with the presence of two N-linked glycosylation consensus sequences located in the propeptide region and not in the mature enzyme region (6, 39). Therefore, we hypothesized that the 35-kDa protein is possibly a N-glycosylated form of LOPP with an unusually high apparent molecular weight due to its unusually high net cationic charge (isoelectric point = ~12). To test our hypothesis, we performed deglycosylation studies on the conditioned media samples from cells treated with TGF-beta1, which is known to increase steady-state lysyl oxidase mRNA levels in a dose- and time-dependent manner in MC3T3-E1 cells (10). MC3T3-E1 cells were cultured in the presence or absence of 400 pM TGF-beta1 for 18 h. Under these conditions, most secreted prolysyl oxidase is processed to mature lysyl oxidase and LOPP (10). Media were harvested and treated with the N-glycosidase PNGase F as described in MATERIALS AND METHODS. Western blot analysis with the LOPP antibody showed the increased presence of a 35-kDa protein in media samples after TGF-beta1 treatment (Fig. 4B, lanes 2 and 4). The apparent molecular mass diminished following treatment with PNGase F, resulting in the same mobility as recombinant nonglycosylated lysyl oxidase propeptide expressed in E. coli (Fig. 4B, lanes 5 and 1). These data indicate that the 35-kDa immunoreactive protein is N-glycosylated LOPP.

LOPP binds tubulin in vitro. Immunofluorescence analyses showed that LOPP colocalizes with microtubules in osteoblasts. We next asked whether LOPP interacts directly with tubulin. To answer this question, we studied the interaction between recombinant LOPP and microtubules using two different approaches. First, we used the far Western blot (tubulin overlay) technique. Ten micrograms of purified LOPP and bovine serum albumin (Sigma) were each subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then overlaid with dimeric tubulin or taxol-promoted polymerized tubulin. Blots were washed, and bound tubulin was then detected with anti-tubulin and peroxidase-coupled secondary antibodies. Data (Fig. 5A) indicate that only recombinant LOPP bound significantly to tubulin, whereas serum albumin binding to tubulin was very weak. Taxol polymerization of tubulin did not appear to promote or inhibit binding to LOPP under these conditions.


Figure 5
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  		Fig. 5. Lysyl oxidase propeptide binds to tubulin in vitro. A: nitrocellulose strips containing electrophoretically separated and transferred recombinant LOPP and control protein and BSA (10 µg) were overlaid with a solution of purified tubulin or polymerized tubulin (10 µg/ml). Bound tubulin was then revealed by immunodetection using anti-tubulin antibody. Lanes 1 and 2 were incubated with taxol-treated (polymerized) tubulin, lanes 3 and 4, with untreated tubulin, and lanes 5 and 6, with vehicle alone. Lanes 1, 3, and 5 were loaded with LOPP; lanes 2, 4, and 6, with BSA. The arrow points to the bound tubulin detected on this far Western blot. The 36-kDa band reflects trace amounts of SDS-resistant aggregates of LOPP, whereas the band running faster than 20 kDa reflects limited proteolysis of LOPP. B: LOPP/nickel affinity column matrix was incubated with day 6 cell extract or cell lysis buffer, or extract was incubated with nickel affinity column without LOPP and washed. Proteins bound to the matrixes were eluted with SDS-PAGE sample buffer, resolved on SDS-PAGE, and analyzed by Western blot analysis using anti-tubulin antibody. Lane 1, elution from the affinity column containing LOPP treated with cell extract; lane 2, elution of affinity column containing LOPP with cell lysis buffer alone (no cell extract); lane 3, affinity column without LOPP plus cell extract. Lane 4 is a sample of the day 6 cell extract before application to the nickel affinity column material showing the presence of tubulin.

 
Additional binding experiments were carried out by using pull-down assays to independently assay for LOPP/tubulin binding. Purified His-tagged LOPP was bound to HIS-Select resin, a nickel affinity matrix, as described under MATERIALS AND METHODS. LOPP binding to the resin was confirmed after elution with sample buffer and Western blot analysis of a small aliquot of the resin treated with LOPP. The LOPP matrix was then incubated with day 6 MC3T3-E1 cell extract, and after extensive washing, the retained proteins were eluted with SDS-PAGE sample buffer and analyzed by Western blot analysis with anti-tubulin antibody. Controls were LOPP matrix incubated with cell lysis buffer, and resin containing no bound LOPP was incubated with the day 6 cell extract. The results showed that only the affinity matrix containing bound LOPP was able to pull down tubulin from day 6 cell extract, in contrast to the controls (Fig. 5B). Together, the binding studies indicate that LOPP binds directly to tubulin in early differentiating MC3T3-E1 cells.

LOPP uptake study. The biosynthetic pathway of lysyl oxidase includes synthesis and secretion of the 50-kDa lysyl oxidase proenzyme, followed by extracellular proteolysis by BMP-1 and related proteases to form active ~30-kDa lysyl oxidase and LOPP. Active lysyl oxidase has been shown to reenter cells, and we postulate that LOPP similarly may reenter cells and exert biological effects. Therefore, we performed a study of the uptake of exogenously added LOPP that contains an NH2-terminal Xpress tag epitope (Invitrogen) that is recognized by a highly specific monoclonal antibody (15). LOPP (10 µg/ml) was added to proliferating MC3T3-E1 cells, and cells were subjected to fixation and immunofluorescence analyses with the anti-Xpress antibody. Colocalization was also determined with tubulin. After 5 min of incubation, punctate cell-associated LOPP was seen (data not shown). By 90 min, intracellular LOPP was more obvious and appeared to be bound to cytoskeletal elements, some of which colocalized with tubulin (Fig. 6). By 150 min, staining was more evenly distributed in the cell and mostly colocalized with tubulin. Thus exogenously added LOPP is taken up and ultimately associates with microtubules, and this resembles the natural association of LOPP with tubulin shown in Fig. 3A.


Figure 6
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  		Fig. 6. Internalization of exogenous LOPP. Subconfluent MC3T3-E1 cells were treated with or without recombinant soluble LOPP/Xpress fusion protein (15) (10 µg/ml) for intervals up to 150 min. The internalization of LOPP and its association with tubulin were evaluated by confocal fluorescence microscopy after samples were stained with mouse anti-Xpress antibody and goat anti-tubulin antibody, followed by rabbit anti-mouse Alexa Fluor 568 and rabbit anti-goat Alexa Fluor 488 conjugates, respectively. Top, single staining of LOPP (red); bottom, double staining of LOPP (red) and tubulin (green). Nuclear staining with Topro-3 (blue) was also conducted at 0 min only to help define cell morphology. An oil objective at x63 was employed; scale bar = 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous studies report that intact LOPP was detected in osteoblastic cell layers (15). In the present study, we examined the subcellular distribution of molecular forms of lysyl oxidase, including LOPP in MC3T3-E1 cells, which are osteoblastic cells derived from newborn mouse calvaria (37). Present data show that forms of lysyl oxidase have a stage-dependent subcellular distribution in osteoblasts. The results obtained from confocal immunofluorescence analyses and Western blot analysis studies demonstrate that LOPP epitopes are mainly localized in the perinuclear region in proliferating MC3T3-E1 cells and have a predominant apparent colocalization with microtubules in differentiating cells. LOPP exists both as the released propeptide resulting from procollagen C-proteinase processing and as part of nonprocessed prolysyl oxidase.

The intracellular localization of LOPP is intriguing. It is known that lysyl oxidase proenzyme is converted into mature lysyl oxidase, releasing propeptide by extracellular procollagen C-proteinases (6, 33, 39, 41). Present data suggest that LOPP is likely taken up by osteoblastic cells after extracellular release. A cellular uptake of LOPP would be feasible based on the unusually high isoelectric point of 12.5 for the mouse, rat, and human LOPP (32). The phenomenon that basic peptides have the ability to pass through cell membranes without the aid of specific receptors has gained increasing biochemical and therapeutic interest (11), and internalization mechanisms, including endocytosis and heparin sulfate proteoglycan binding, have been shown to be involved (2, 27). This is the mechanistic basis for the use of basic TAT peptide sequences to promote cell uptake of recombinant fusion proteins (40). LOPP contains a putative nuclear localization signal, PQRRRDS (residues 39–45), as shown by the PSORT program (26). Li et al. (25) pointed out that residues 41–60 of rat LOPP, RRRDSATAPRADGNAAAQPR, is similar to the nuclear localization signal identified in N-myc, ERRRNHNILERQRRND (4). Thus, once inside cells, LOPP could be directed into the nucleus by the nuclear localization signal. In addition, internalized LOPP would be available to bind tubulin and microtubules in the cytosol, as shown by new data provided in this report. We speculate that the lack of apparent coassociation of LOPP with microtubules in proliferating osteoblasts as shown in Fig. 1 is due to insufficient production and accumulation of extracellular LOPP that ultimately reenters cells. This notion is consistent with previous findings that show increased accumulation of LOPP in differentiating osteoblasts (15) and with the LOPP uptake data presented in Fig. 6.

The present study detected forms of LOPP with apparent molecular weights of 35,000 and 18,000, both of which are considerably higher than the predicted molecular weight of 15,290 of the nonglycosylated mouse LOPP following signal peptide removal and procollagen C-proteinase processing. Studies with PNGase F demonstrated that the 35-kDa LOPP is N-glycosylated, and the resulting deglycosylated polypeptide matches the mobility of the 18-kDa band. It is known that the 50-kDa lysyl oxidase proenzyme is N-glycosylated and that the two consensus N-glycosylation sites are contained within the propeptide region (6, 33, 39). The apparent molecular weight of the 35-kDa LOPP is much higher than predicted, given that each N-glycosylation should result in an increase of ~1.5–3.0 kDa each, depending on whether the N-linked carbohydrates are high mannose, hybrid, or complex. In addition, it is notable that the apparent molecular weights of both the nonglycoyslated and glycosylated LOPP on SDS-PAGE are higher than predicted. The unexpected slow mobility of these proteins is very likely due to the highly basic character of LOPP. Proteins containing highly basic regions exhibit similarly slow mobility on SDS-PAGE (16), and posttranslational modifications of basic proteins result in changes in mobility that are larger than predicted (18).

In the present study, we found that LOPP interacts and binds to tubulin in differentiating osteoblasts. Microtubule-associated proteins (MAPs) are proteins that bind to tubulin via electrostatic interactions (7) and modulate the assembly and the stability of microtubules and mediate their interactions with other cellular components (29, 43). Such charge-charge interactions may also account for the specific binding between the LOPP and negatively charged microtubules reported presently. Microtubules are cytoskeletal structures that are critical for various cellular functions such as the determination of cell division, cell shape and polarity, cell movement, intracellular transport, and signal transduction (7, 8, 12, 13). The association between LOPP and microtubules may suggest potential functions of LOPP in controlling osteoblast proliferation or differentiation. It will be interesting to determine in future studies whether LOPP and lysyl oxidase play synergistic or opposing functional roles in developing osteoblasts.

It also is of interest that prolysyl oxidase and mature lysyl oxidase were found inside osteoblasts and that lysyl oxidase, like LOPP, was found to be associated with microtubules in differentiating cells. Filamentous staining of lysyl oxidase resembling cytoskeletal association was reported in skin fibroblasts some time ago, detected with a highly specific monoclonal antibody against mature lysyl oxidase (24, 42). In smooth muscle cells and NIH/3T3 fibroblasts, mature lysyl oxidase was found in the nucleus (25). Studies have shown that mature lysyl oxidase is taken up by smooth muscle cells and then translocated to the nucleus (28). At this time, intracellular functions for mature lysyl oxidase are not known. The present study clearly identified the presence of prolysyl oxidase in the nuclei of proliferating osteoblasts, and at this time it is not known whether this occurs by a reuptake mechanism or is derived from a proenzyme pool that is not secreted from osteoblasts. The elucidation of the trafficking and functional role of intracellular prolysyl oxidase and mature lysyl oxidase will be of great interest.

In summary, the present study provides new insights into the production and localization of molecular forms of lysyl oxidase in osteoblasts. Data are consistent with a model in which LOPP is generated extracellularly by proteolytic processing by procollagen C-proteinases, taken up by osteoblasts, and distributed principally to microtubules and to the nucleus. Future studies must examine whether the intracellular localization of LOPP has functional consequences in osteoblastic cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by National Institute of Dental Research Grants DE-12209, DE-14066, and DE-11004 and a research fellowship from the Deutsche Forschungsgemeinschaft, Germany.


   ACKNOWLEDGMENTS
 
We thank Dr. Patima Sdek and Dr. Shenghe Cai for valuable discussions and suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. C. Trackman, Division of Oral Biology, Boston Univ. Goldman School of Dental Medicine, 700 Albany St., Boston, MA 02118 (e-mail: trackman{at}bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Y. Guo and N. Pischon contributed equally to this work. Back


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 DISCUSSION
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