©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pre- and Post-translational Regulation of Lysyl Oxidase by Transforming Growth Factor-1 in Osteoblastic MC3T3-E1 Cells (*)

(Received for publication, August 15, 1995; and in revised form, October 18, 1995)

Eduardo J. Feres-Filho (1) Young Jin Choi (1) Xiaoyan Han (3) Timo E. S. Takala (3) Philip C. Trackman (1) (2)(§)

From the  (1)Department of Periodontology and Oral Biology, Boston University Goldman School of Graduate Dentistry, Boston, Masachusetts, the (2)Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, and the (3)Department of Biology of Physical Activity, University of Jyväskylä, Jyväskylä, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The final enzymatic step required for collagen cross-linking is the extracellular oxidative deamination of peptidyl-lysine and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous extracellular matrix is required for bone formation. The goals of this study were to compare the transforming growth factor (TGF)-beta1 regulation of lysyl oxidase enzyme activity and steady state mRNA levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-beta1 increased steady state lysyl oxidase and COL1A1 mRNA levels in a dose- and time-dependent manner. The increase in lysyl oxidase mRNA levels was transient, peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-beta1. COL1A1 steady state mRNA levels increased maximally to 3.5-fold of controls. Development of increased lysyl oxidase enzyme activity was delayed and was of slightly lower magnitude than the increase in its mRNA levels. This suggested limiting post-translational processing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme secretion and proteolytic processing. Development and application of an independent assay for lysyl oxidase proenzyme proteolytic processing activity verified its proportionately lower stimulation by 400 pM TGF-beta1. Thus, lysyl oxidase regulation by TGF-beta1 in osteoblastic cell cultures occurs at both pre- and post-translational levels. This regulation is consistent with increased production of a collagenous extracellular matrix.


INTRODUCTION

Lysyl oxidase is the extracellular enzyme that catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin precursors to form peptidyl-alpha-aminoadipic--semialdehyde, or peptidyl--hydroxy-alpha-aminoadipic--semialdehyde, respectively. Spontaneous condensation reactions of these resultant aldehydes leads to the formation of lysine-derived cross-links found in mature collagens and elastin(1) .

A cross-linked collagen matrix is required for differentiation of osteoblastic cells and for bone mineralization(2) . Type I collagen synthesis and accumulation have been shown to be uncoupled processes in developing MC3T3-E1 osteoblastic cells(3) . These cells accumulated collagen in the extracellular matrix when the rate of collagen synthesis was decreasing. This suggested post-translational control of collagen fibril accumulation. Similarly, in primary rat calvaria osteoblast cell cultures, TGF-beta1(^1)-stimulated extracellular collagen accumulation was controlled by unidentified post-translational steps(4) . Lysyl oxidase-mediated control of collagen accumulation in bone cell cultures has been described(5) . In this study, treatment of chick osteoblast cultures with the lysyl oxidase inhibitor, beta-aminopropionitrile (BAPN), reduced the accumulation of collagen in the cell layer by 50%. Turnover of collagen deposited into the extracellular matrix was also markedly increased in BAPN-treated cultures. Further evidence for the importance of lysyl oxidase in bone formation was illustrated by in vivo studies, where growing chicks and rats fed with BAPN developed skeletal deformities known as osteolathyrism(6, 7) .

TGF-beta increased accumulation of extracellular matrix proteins in different cell and organ culture models(4, 8, 9, 10) . In vivo, the highest levels of TGF-beta were found in platelets and bone(11, 12) . TGF-beta-mediated inhibition of osteoblastic cell growth was accompanied by increased synthesis of type I collagen, fibronectin, and proteoglycans(13, 14, 15) . TGF-beta also decreased the expression of extracellular matrix degrading enzymes, and induced genes for protease inhibitors in different cell types(16, 17) . Among the genes that were induced by TGF-beta in osteoblastic MC3T3-E1 cells, steady-state lysyl oxidase mRNA levels increased almost 10-fold(18) .

Although lysyl oxidase enzyme activity was not measured in the studies cited above, taken together they raise the possibility that like collagens, lysyl oxidase is a major target for TGF-beta in osteoblasts. If true, then lysyl oxidase regulation by TGF-beta could contribute to increased collagen accumulation by osteoblastic cells stimulated by this growth factor.

The biosynthesis of lysyl oxidase is complex and requires numerous post-translational modifications, each of which could potentially control activation of lysyl oxidase enzyme activity. Lysyl oxidase is synthesized as a 50-kDa glycoprotein precursor, and is then secreted and proteolytically processed to the mature 32-kDa enzyme(19) . Recently, the cleavage site of the NH(2)-terminal propeptide region of the lysyl oxidase precursor was reported(20) . Lysyl oxidase is a copper metalloenzyme, and in addition contains an organic cofactor believed to be a quinone derived from post-translational modification of a tyrosine residue(1, 21) . The mechanisms of post-translational constitution of the metal and organic cofactors with lysyl oxidase proenzyme have not yet been defined.

In the present study, we report that TGF-beta1 dramatically increased the levels of lysyl oxidase mRNA and enzyme activity in MC3T3-E1 osteoblastic cultures. The effects on steady-state lysyl oxidase and COL1A1 mRNA levels were compared. Furthermore, we found that post-translational mechanisms may control the expression of fully active lysyl oxidase.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human transforming growth factor beta-1 (TGF-beta1) was purchased from Austral Biologicals, San Ramon, CA. TRI Reagent LS was obtained from Molecular Research Center, Inc., Cincinnati, OH. Dulbecco's modified Eagle's medium, newborn bovine serum, trypsin-EDTA solution, penicillin-streptomycin solution, Dulbecco's phosphate-buffered saline, non-essential amino acids solution, bovine albumin fraction V, ascorbic acid, and beta-glycerophosphate were purchased from Sigma. The Protein Fusion and Purification System was purchased from New England Biolabs, Beverly, MA. All other chemicals were of reagent grade.

Cell Culture

Murine osteoblast-like MC3T3-E1 cells were provided by Dr. Louis Gerstenfeld, Children's Hospital, Boston, MA, and Dr. Renny Franceschi, University of Michigan, Ann Arbor. Cells were plated onto 100-mm tissue culture dishes in Dulbecco's modified Eagle's medium, containing 10% heat-treated (56 °C, 30 min) newborn calf serum plus 1% non-essential amino acids and 100 µg/ml of each penicillin and streptomycin. Cultures were maintained at 37 °C in a fully humidified atmosphere of 5% CO(2) in air. Media were changed every 3 days. Cells in the logarithmic growth phase were dissociated with trypsin/EDTA, and inoculated at 200,000 cells/plate. After 2.5 days the subconfluent cells were then fed with serum-free medium, containing 0.1% bovine serum albumin, 50 µg/ml ascorbate, and 10 mM beta-glycerophosphate and cultured for an additional 24 h. Cells were then refed with fresh media plus or minus TGF-beta1, for the appropriate period of time. Experiments were performed after no more than 3 passages from one set of frozen cell stocks.

Assay of Lysyl Oxidase Activity

Lysyl oxidase enzyme activity was measured in the conditioned media and cell layers, using recombinant human [^3H]tropoelastin substrate (22) . Media samples (0.5 ml) were assayed in quadruplicate in a final volume of 1 ml, containing 0.1 M borate, 0.15 M NaCl, pH 8.0, and 160,000 cpm of [^3H]tropoelastin in the presence and absence of 5 times 10M BAPN. Reactions were incubated for 90 min at 37 °C, followed by distillation under vacuum. Radioactivity in 0.5-ml aliquots of distillate was determined by liquid scintillation spectrometry. Units of enzyme activity were defined as dpm released above the BAPN control. Enzyme activity was normalized to cell number, after dissociating cells from the same plate with trypsin/EDTA, and counted using a hemocytometer.

RNA Isolation and Northern Analysis

Total RNA was prepared using Tri-Reagent LS(23) . Ten micrograms of denatured RNA was applied per lane and separated by electrophoresis on 1% agarose gels, containing 18% formaldehyde, and transferred to GeneScreen (DuPont) nylon membranes using 10-fold SSC (1 times SSC, 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Membranes were baked at 80 °C for 1 h and prehybridized at 42 °C for 4 h in hybridizing solution (50% formamide, 1-fold SSPE (1 times SSPE, 0.15 M NaCl, 0.01 M NaH(2)PO(4), 0.02 M EDTA, pH 7.4), 10% 5-fold Denhardt's solution, 0.2% SDS, 0.05% Na(2)PP(i) and 1% salmon sperm DNA). Membranes were then hybridized for 18 h at 42 °C with a P-labeled COL1A1 cDNA (24) or P-labeled mouse lysyl oxidase cDNA probe (25) . For normalization, blots were stripped and rehybridized with a radiolabeled 18 S ribosomal probe(26) . 1 times 10^6 cpm of probe was used per ml of hybridizing solution. Probes were labeled using the random primer method(27) . The membranes were washed as described and subjected to autoradiography(28) . Autoradiograms were assessed and normalized by densitometric scanning. Values for standard error were derived from triplicate scans of films. Experiments were performed at least twice.

Pulse Labeling and Immunoprecipitation

Cells were grown as described above and then fed with serum free medium ± TGF-beta1 (400 pM) for the appropriate period of time. Cultures were then refed and incubated for 20 min with 7 ml/plate of serum-free and methionine-free Dulbecco's modified Eagle's medium ± TGF-beta1 (400 pM). Cultures were then placed in fresh media ± TGF-beta1 (400 pM) supplemented with 50 µCi/ml [S]methionine (1175 Ci/mmol; DuPont NEN, catalog number NEG-072). Following incubation, media and cell layers were harvested and prepared for immunoprecipitation(19) . Constant counts/min from the cell layer, and medium were used for immunoprecipitation with rabbit anti-bovine aortic lysyl oxidase(19) . Samples were subjected to SDS-PAGE, and the gels were treated with Resolution (EM Corp., Chestnut Hill, MA) and dried under vacuum. Gels were placed on Kodak XAR-5 film with an intensifying screen at -80 °C for 5-30 days.

Rat Lysyl Oxidase Fusion Protein Production and Purification from Escherichia coli

A bacterial expression vector was constructed utilizing rat lysyl oxidase cDNA (28, 29) cloned downstream and in-frame with the malE gene of E. coli in vector pMAL-c2(30, 31) . The construct encodes the lysyl oxidase proenzyme beginning at amino acid residue 25 cloned into the XmnI site of pMAL-c2. The cloning strategy resulted in introduction of no non-lysyl oxidase residues into the vector. Although not relevant to the present study, if the fusion protein were cleaved by Factor Xa as designed, the resultant 43,748-Da lysyl oxidase proenzyme would begin four amino acid residues to the carboxyl side of the predicted signal peptide cleavage site(28) .

The lysyl oxidase construct was cloned in three steps. Fragment I was lysyl oxidase coding region from bp 42 to bp 233 derived from a PstI fragment cloned into pUC 18 vector as described below (28, 29) . Fragment II was lysyl oxidase coding region from bp 234 to bp 939 (PstI/HindIII fragment). Fragment III was a HindIII fragment containing lysyl oxidase coding region from bp 940 through the end of the coding region, and extending to the first EcoRI site in the 3`-untranslated region. Fragments II and III were derived from the lysyl oxidase transcription vector pBSCOD (19) .

pUC 18 containing fragment I was digested with Eco0109I and recessed 3` termini were filled in using the Klenow fragment of E. coli DNA polymerase. Then the fragment was digested with PstI, yielding the 230-bp blunt/PstI fragment I. pBSCOD containing Fragments II and III was digested with PstI/HindIII, yielding Fragment II. This DNA was cloned into a PstI/HindIII-digested pMAL-p2 vector. pMAL-p2 DNA containing Fragment II was isolated from positive clones, digested with Xmn I/PstI, and ligated with blunt/PstI Fragment I, and correct clones were isolated. This pMAL-p2 plasmid containing Fragments I and II was digested with HindIII and 600-bp HindIII/HindIII Fragment III was cloned into this site. The resulting construct was lysyl oxidase cloned into pMAL-p2 (pLO/P2). As fusion protein expression from pLO/P2 was unacceptably low, we then recloned the NdeI/NcoI fragment of LO/P2 into the NdeI/NcoI site of pMAL-c2 resulting in construct pLO/C2. Limited DNA sequence analyses of these constructs revealed the correct nucleotide sequences at the junctions of lysyl oxidase Fragments I, II, and III. Detailed restriction mapping also confirmed the structure of these constructs.

pLO/C2 was used to transform E. coli strain BL21 (DE3)pLys(S) (Novagen, Inc). Two liters of logarithmic phase cells were induced with 0.3 mM 5-bromo-4-chloro-3-indoyl-beta-D-glucopyranoside for 4.5 h, collected by centrifugation at 5000 times g, and stored at -80 °C. The pellet was thawed and resuspended in 200 ml of 50 mM Tris, 2 mM EDTA, pH 8.0. At room temperature, 1.2 ml of 0.2 M phenylmethylsulfonyl fluoride, 25 ml of 10 mM MgCl(2), and 10 mg of protease-free DNase I dissolved in 8 ml of 0.15 M NaCl were added to the lysed cell pellet and stirred for 10 min. The inclusion bodies were collected by centrifugation, and then solubilized in 280 ml of 8 M urea denaturing buffer, pH 7.8 (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8), and dialyzed against 10 liters of 1 M urea denaturing buffer, pH 7.8, at 4 °C overnight, followed by several changes of amylose column buffer (20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA, 10 mM beta-mercaptoethanol) over 24 h. The soluble proteins were then applied to an amylose column (New England Biolabs) as described(31) . The fusion protein was eluted with column buffer containing 10 mM maltose, with a typical yield of 6.4 mg from 2 liters of culture. The eluted fusion protein was further purified by chromatography on hydroxylapatite as follows. Nine mg of fusion protein dialyzed against 10 mM KH(2)PO(4), pH 7.2, was applied to a 10-ml hydroxylapatite column (Sigma) equilibrated in the same buffer. The column was successively washed with 40 ml of 10 mM KH(2)PO(4), pH 7.2, 40 ml of 500 mM KH(2)PO(4), pH 7.2. The column was then eluted with 6 M urea, 20 mM KH(2)PO(4), pH 7.2. The yield was 3.21 mg or 35%. The purified fusion protein was exhaustively dialyzed into 50 mM Tris, pH 7.8, prior to use. Purity was assessed by SDS-PAGE.

NH(2)-terminal Analysis of Cleaved Lysyl Oxidase Fusion Protein

MC3T3-E1 cells were treated with 400 pM TGF-beta1 in serum-free media containing 0.01% bovine serum albumin, ascorbate, and beta-glycerol phosphate for 24 h. Three ml of medium was then mixed with 180 µg of fusion protein (3.5 ml in 50 mM Tris, pH 7.8) and incubated at 37 °C for 3 h (final volume of 6.5 ml). Proteins were precipitated overnight by adding trichloroacetic acid to 10% after cooling sample to 4 °C, collected by centrifuging at 10,000 times g, washed twice with cold acetone, and then subjected to 10% SDS-PAGE. Sample was electroblotted onto polyvinylidene difluoride membranes (Bio-Rad) and stained with Ponceau S (Sigma). The 32-kDa band was excised and submitted to the Harvard Microchemistry Facility, Cambridge, MA, for NH(2)-terminal sequence analysis.


RESULTS

Dose-dependent Up-regulation of Lysyl Oxidase by TGF-beta1

Preconfluent MC3T3-E1 cells were cultured in serum-free media supplemented with 0-400 pM TGF-beta1 for 24 h. A dose-dependent up-regulation in the steady-state lysyl oxidase and COL1A1 mRNA levels was found (Fig. 1). Lysyl oxidase mRNA increased 8.8-fold in the presence of 400 pM TGF-beta1. COL1A1 steady-state mRNA levels also increased but reached a modest level of 3.5-fold of control at 400 pM TGF-beta1. Lysyl oxidase enzyme activity in the cell culture media was stimulated by 4 and 40 pM TGF-beta1 comparable to increases observed in the mRNA levels (Table 1). In contrast, 400 pM TGF-beta1 caused a smaller increase in lysyl oxidase enzyme activity (5.4-fold) compared to the 8.8-fold increase in lysyl oxidase steady-state mRNA levels. No enzyme activity was detected in the cell layers (data not shown). These experiments were performed twice yielding similar results.


Figure 1: Dose-dependent regulation of lysyl oxidase and COL1A1 steady-state mRNA levels by TGF-beta1. MC3T3-E1 cells were cultured in the presence of TGF-beta1 (0, 4, 40, and 400 pM). After 24 h, total RNA was extracted and subjected to Northern blotting. Inset, autoradiograms of Northern blots probed with P radiolabeled lysyl oxidase and COL1A1 cDNAs, and with DNA for 18 S ribosomal RNA (18S rRNA). Lane 1, 0 TGF-beta1; lane 2, 4 pM TGF-beta1; lane 3, 40 pM TGF-beta1; lane 4, 400 pM TGF-beta1. Black bars, lysyl oxidase; hatched bars, COL1A1. Signals were quantitated by scanning laser densitometry and were normalized to 18 S rRNA. Values represent mean ± S.D. obtained from three scanning laser densitomentry determinations from one representative experiment.





Time-dependent Regulation of Lysyl Oxidase

The time-dependent regulation of steady-state lysyl oxidase mRNA levels and enzyme activity by 400 pM TGF-beta1 was determined. TGF-beta1 increased steady-state lysyl oxidase mRNA levels after 3 h of treatment, reaching a maximum at 12 h ( Fig. 2B, lanes 5 and 6). Lysyl oxidase enzyme activity in the cell culture media was stimulated to a lesser degree, and peaked later, at 18 h (Fig. 2A). The delay between steady-state lysyl oxidase mRNA levels and enzyme activity suggests that post-translational mechanisms may limit the formation of fully active lysyl oxidase. The following experiments were performed to investigate potential limiting steps in the biosynthesis of fully active lysyl oxidase.


Figure 2: Time-dependent effect of 400 pM TGF-beta1 on lysyl oxidase enzyme activity (A); and steady-state levels of lysyl oxidase and COL1A1 mRNAs (B) from parallel cultures. A, lysyl oxidase was measured in the conditioned medium, using recombinant human [^3H]tropoelastin substrate. Values are mean ± S.E. of samples assayed in quadruplicate and normalized to 10^6 cells counted from the same plate of cultured cells. B, autoradiograms of Northern blots of total RNA (10 µg/lane) from MC3T3-E1 cells cultured in parallel in the absence (odd numbered lanes) or presence (even numbered lanes) of 400 pM TGF-beta1. Lanes 1 and 2, 3 h; lanes 3 and 4, 6 h; lanes 5 and 6, 12 h; lanes 7 and 8, 18 h; lanes 9 and 10, 21 h; lanes 11 and 12, 24 h. Data are from one of two replicate experiments.



Synthesis and Secretion of the Lysyl Oxidase Precursor

We tested whether TGF-beta1 increased the synthesis of lysyl oxidase precursor protein parallel to changes in steady-state lysyl oxidase mRNA. Cells were pretreated with or without 400 pM TGF-beta1 for 4 or 15 h, and then pulse labeled with [S]methionine for 3 h. Radioactive media and extracts of cell layers were then immunoprecipitated with anti-bovine lysyl oxidase as described previously(19) . Fig. 3shows autoradiograms of lysyl oxidase immunoprecipitates from the media (lanes 1-5) and cell layers (lanes 6-9) of these cultures. TGF-beta1 increased the labeled pool of 50 ± 5 kDa lysyl oxidase precursor in the cell layer after 4 and 15 h of growth factor pretreatment compared to control cultures (Fig. 3, lanes 7 and 9). A 5.5-fold increase in the amount of labeled proenzyme was detected after 15 h of growth factor pretreatment compared to 4 h of pretreatment, revealed by scanning laser densitometry of autoradiograms. This change was similar to that seen at the mRNA level (Fig. 2B), where a 6.9-fold increase in lysyl oxidase steady state mRNA levels occurred between 3 and 12 h of treatment with TGF-beta1. Thus, as expected, increases in cell layer proenzyme labeling closely paralleled changes in lysyl oxidase steady-state mRNA. Under these conditions of pulse-labeling, no lysyl oxidase proenzyme was detected in unstimulated control cultures (Fig. 3, lanes 6 and 8).


Figure 3: Short-term pulse labeling/immunoprecipitation/SDS-PAGE of lysyl oxidase proteins from MC3T3-E1 cells pretreated with TGF-beta1 for 4 or 15 h. MC3T3-E1 cells were pretreated without (control) or with 400 pM TGF-beta1 for 4 or 15 h, and then pulse-labeled with [S]methionine for 3 h. Constant cpm of media proteins (7 times 10^6 cpm) and cell layer extracts (2 times 10^7 cpm) were immunoprecipitated with anti-lysyl oxidase antibody. Samples were then subjected to SDS-PAGE, and autoradiography. Autoradiograms of immunoprecipitates from media (lanes 1-5): lane 1, control (4 h); lane 2, TGF-beta1 (4 h); lane 3, control (15 h); lane 4, TGF-beta1 (15 h); lane 5, as in lane 4 except immunoprecipitation was performed in the presence of 50 µg of unlabeled lysyl oxidase/maltose-binding fusion protein. Immunoprecipitates from the cell layer (lanes 6-9): lane 6, control (4 h); lane 7, TGF-beta1 (4 h); lane 8, control (15 h); lane 9, TGF-beta1(15 h). Molecular weight markers were Bio-Rad prestained low molecular weight range markers.



Levels of radioactive secreted proenzyme from growth factor pretreated cells were slightly lower after 15 h compared to 4 h under these conditions of pulse labeling (Fig. 3, lanes 2 and 4). Control media had no detectable radiolabeled proenzyme (Fig. 3, lanes 1 and 3). No fully processed labeled 32-kDa lysyl oxidase was detected under these conditions in control or growth factor pretreated cultures (Fig. 3, lanes 1-4). Consistent with previous studies(19) , the immunoprecipitations were shown to be specific (Fig. 3, lane 5). These findings suggest that secretion and extracellular proteolytic processing of lysyl oxidase requires more than 3 h in MC3T3-E1 cells. Thus, due to a large unlabeled intracellular lysyl oxidase pool after 15 h of TGF-beta1 pretreatment, secreted proenzyme was likely of lower specific radioactivity than that secreted from cells pretreated for only 4 h. This would account for the lower labeling of secreted proenzyme at 15 h compared to 4 h of growth factor pretreatment.

Accumulation of Media Lysyl Oxidase Proenzyme in TGF-beta1-stimulated Cultures

We next tested whether proteolytic processing may be insufficient to fully convert the lysyl oxidase proenzyme secreted from TGF-beta1-stimulated cells. Cells were treated with or without 400 pM TGF-beta1 for 12 and 18 h in the continuous presence of radioactive methionine. Media and cell layer extracts were then immunoprecipitated with lysyl oxidase antibodies. This experiment revealed the molecular forms of lysyl oxidase that accumulate in control and growth factor-treated cell cultures over relatively long periods of pulse labeling.

As shown in Fig. 4(lanes 1-4), TGF-beta1 increased the pool of lysyl oxidase precursor in the conditioned media in a time-dependent manner. Similarly, a greater amount of labeled 32-kDa lysyl oxidase was found at 18 h compared to 12 h. This is consistent with increased enzyme activity observed after 18 h of growth factor treatment (Fig. 2). Densitometric scanning of autoradiograms from 12- and 18-h immunoprecipitates showed 3-fold increases in the 50 ± 5 kDa precursor (12 h, 689 ± 5.6; 18 h, 1, 964 ± 25.5) and in the 32 ± 3 kDa form of lysyl oxidase (12 h, 378 ± 33.2; 18 h, 1, 121 ± 29.7) in TGF-beta1-treated cells. The accumulation of 50 ± 5-kDa lysyl oxidase only in the media of TGF-beta1-treated cultures suggests that the conversion to the 32 ± 3-kDa species was limited by extracellular proteolytic processing activity.


Figure 4: Continuous pulse-labeling/immunoprecipitation/SDS-PAGE of lysyl oxidase proteins from MC3T3-E1 cells treated with TGF-beta1. MC3T3-E1 cells were treated without (control) or with 400 pM TGF-beta1 in the presence of [S]methionine continuously for 12 and 18 h. Constant cpm of media proteins (3 times 10^7 cpm) and cell layer extracts (1 times 10^8 cpm) were immunoprecipitated with anti-lysyl oxidase antibody. Samples were subjected to SDS-PAGE. Autoradiograms of immunoprecipitates from the media (lanes 1-4): lane 1, control (12 h); lane 2, TGF-beta1 (12 h); lane 3, control (18 h); lane 4, TGF-beta1 (18 h). Immunoprecipitates from the cell layers (lanes 5-8): lane 5, control (12 h); lane 6, TGF-beta1 (12 h); lane 7, control (18 h); lane 8, TGF-beta1 (18 h).



Scanning laser densitometry of Fig. 4, lanes 1-4, revealed that the level of labeled 32 ± 3-kDa lysyl oxidase increased by 13- and 20-fold in TGF-beta1-treated cells compared to unstimulated cells at 12 and 18 h, respectively. Assuming that the 32 ± 3-kDa molecular form of lysyl oxidase was fully active and that the specific radioactivity of methionine pools was comparable in these continuously labeled cultures, corresponding increases in lysyl oxidase enzyme activity by TGF-beta1 of 10-20-fold would be expected. Interestingly, we observed at most only a 6-fold stimulation of enzyme activity at 18 or 24 h ( Table 1and Fig. 2). We conclude that lysyl oxidase is unlikely to be fully active in TGF-beta1-stimulated cultures (see ``Discussion'').

Results obtained from the cell layer suggest that relatively low levels of lysyl oxidase proenzyme accumulation occurred in this fraction compared to the media (Fig. 4). Moreover, absence of the 32 ± 3-kDa molecular form in the cell layer correlated well with the absence of enzyme activity. Thus, in these MC3T3-E1 osteoblastic cell cultures, lysyl oxidase was synthesized as a 50 ± 5-kDa precursor (Fig. 3), secreted and processed proteolytically to a 32 ± 3-kDa molecular species that remained soluble (Fig. 4). This is distinct from studies in neonatal rat aorta smooth muscle cells, and rat lung fibroblasts, where lysyl oxidase enzyme activity and the 32 ± 3-kDa molecular form were shown to accumulate in the cell layer after extracellular proteolytic processing(19, 32) .

Extracellular Proteolytic Conversion of Lysyl Oxidase Precursor to the 32-kDa Molecular Form

An independent assay for lysyl oxidase processing proteinases was developed in order to verify results found in pulse-labeling studies suggesting limiting levels in TGF-beta1-stimulated cultures. We produced the lysyl oxidase proenzyme as a fusion protein with the maltose-binding protein in E. coli (see ``Materials and Methods'' and Fig. 5). This 86-kDa fusion protein was used as the substrate to assay for production of a 32 ± 3-kDa protein immunoreactive with lysyl oxidase antibodies. To validate this assay, we also established the NH(2)-terminal amino acid sequence of the immunoreactive cleavage product generated from the fusion protein by MC3T3-E1 conditioned media (see below).


Figure 5: Coomassie Blue-stained SDS-PAGE gel of lysyl oxidase/maltose binding fusion protein. Lane 1, Bio-Rad low molecular weight prestained markers; lane 2, 5 µg of partially purified fusion protein eluted from the amylose affinity column; lane 3, 1.5 µg of purified fusion protein eluted from hydroxylapatite.



Media obtained from MC3T3-E1 cells treated for 24 h with 400 pM TGF-beta1 was incubated with fusion protein in the presence and absence of 20 mM EDTA for up to 3 h, and then analyzed by Western blotting. As shown (Fig. 6) MC3T3-E1 media converted the fusion protein to a discrete 32 kDa ± 3-kDa molecular species clearly immunoreactive with lysyl oxidase antibody in a time-dependent manner. This activity was fully inhibited by 20 mM EDTA, suggesting that this activity is a metalloenzyme (Fig. 6, lane 8). Conversion to 32 kDa ± 3-kDa product was linear with respect to time up to 50% conversion (Fig. 6). If reactions were carried out beyond 50% conversion, production of 32-kDa product as a function of time was no longer linear (Fig. 6, lanes 6 and 7 in inset were not plotted). Production of 32 ± 3-kDa product was also linear with respect to volume of culture medium up to 50% conversion (not shown). Thus, linearity was maintained up to 50% conversion with respect to time of incubation, and volume of MC3T3-E1 culture media. It is likely that loss of linearity beyond 50% conversion was due to substrate depletion or product inhibition. 1,10-Phenanthroline (1 mM) inhibited production of 32 ± 3-kDa product by 87% (Fig. 6, lane 9). This further supports that lysyl oxidase processing activity is a metalloenzyme(s).


Figure 6: Linear time-dependent production of 32-kDa immunoreactive lysyl oxidase from lysyl oxidase/maltose-binding fusion protein substrate by conditioned MC3T3-E1 cell culture medium. MC3T3-E1 cells were treated with 400 pM TGF-beta for 24 h, and 200-µl aliquots of conditioned medium were incubated at 37 °C with 13 µg of purified fusion protein in a final volume of 0.5 ml of 50 mM Tris, pH 7.8. Reactions were stopped at intervals by addition of 1 volume of SDS-PAGE sample buffer, and boiled for 3 min. Fifty-microliter aliquots were subjected to Western blotting and scanning laser densitometry. Inset, Western blot stained with rabbit anti-bovine lysyl oxidase and alkaline phosphatase-coupled goat anti-rabbit IgG visualized with nitro blue tetrazolium/5-borom-4-chloro-3-indoyly phosphate Western Blue substrate (Promega)(28, 40) . Lane 1, zero time; lane 2, 20 min; lane 3, 40 min; lane 4, 60 min; lane 5, 90 min; lane 6, 120 min; lane 7, 180 min; lane 8, same as lane 7 except incubations also contained 20 mM EDTA; lane 9, same as lane 7 except that incubations contained 1 mM 1,10-phenanthroline. The diffuse 65-kDa band is a nonspecific artifact previously noted(40) . Replicate scans did not vary by more than 5% conversion. Markers were Bio-Rad prestained low molecular weight standards.



NH(2)-terminal analysis was performed on the 32 ± 3-kDa product generated by culture media from TGF-beta1-stimulated cells (see ``Methods and Materials'' for details). As shown in Table 2, the analysis is consistent with the sequence Asp-Asp-Pro-Tyr-Ser. This is the same NH(2)-terminal sequence identified for active lysyl oxidase(20) . This sequence does not occur in the maltose-binding protein. Thus, the Western blot assay detects activities that result in cleavage of the lysyl oxidase fusion protein at the correct site.



Processing protease activity as a function of time of TGF-beta1 treatment of MC3T3-E1 cells was determined under valid assay conditions established above. These results were compared to changes in lysyl oxidase enzyme activity in the same media. Lysyl oxidase enzyme activity and processing protease activity were not changed by 400 pM TGF-beta1 after 3-6 h of treatment (Fig. 7). In contrast, from 12 h, both activities were higher in media from growth factor-treated cultures. Interestingly, the maximum increase in lysyl oxidase enzyme activity was greater than the processing protease. Thus, at 15 h, lysyl oxidase activity was 3.4-fold of control levels; whereas processing activity was increased 1.7-2-fold. These results demonstrate that increased lysyl oxidase enzyme activity induced by TGF-beta1 was accompanied by increased proteolytic processing activity. The degree of stimulation of proteolytic processing activity by TGF-beta1 was less than the degree of stimulation of lysyl oxidase enzyme activity. These results support the concept that proteolytic processing of lysyl oxidase may partially limit the production of fully active 32 ± 3-kDa lysyl oxidase in these osteoblastic cell cultures.


Figure 7: Time study of lysyl oxidase processing activity (proteinase) and lysyl oxidase enzyme activity in MC3T3-E1 cells treated with no TGF-beta1 (closed circles) or with 400 pM TGFbeta1 (open boxes). MC3T3-E1 cells were treated with 0 or 400 pM TGF-beta1 as described under ``Methods and Materials.'' Plates were harvested at intervals, and proteinase (see Fig. 6) and lysyl oxidase enzyme activities were determined from the conditioned media, normalized to cell number determined from the same plate. Values for lysyl oxidase activity are means ± S.E. of quadruplicate assays. Values for processing proteinase activity are means of duplicate determinations ± S.E. The volume of media assayed for processing proteinase activity was 125 µl. This enabled all assays to be in the linear range.




DISCUSSION

TGF-beta1 is present in high amounts in bone(12) , and has been shown to stimulate the synthesis of type I collagen, fibronectin, and proteoglycans by osteoblastic cells(14, 15) . Type I collagen synthesis and accumulation have been shown to be uncoupled processes in developing MC3T3-E1 cells(3) . Moreover, TGF-beta1-stimulated collagen accumulation in cell layers of rat calvaria osteoblastic cultures was found to be partially controlled by unidentified post-translational mechanisms(4) . In a separate study, lysyl oxidase mRNA was found to increase 8-fold in MC3T3-E1 cells treated with TGF-beta1(18) . Recently, lysyl oxidase was implicated in regulating collagen accumulation, and in decreasing collagen turnover in mineralizing chick osteoblastic cultures(5) . It was suggested that collagen cross-linking increases irreversible retention of collagen fibrils in the extracellular matrix. This would amplify the efficiency of collagen insolubilization, reduce the pool of soluble collagen, and thereby decrease collagen susceptibility to proteolytic degradation.

In the present study, we investigated whether lysyl oxidase, like COL1A1 is a major target for TGF-beta1 in MC3T3-E1 osteoblastic cultures. We now report that TGF-beta1 significantly increased steady state lysyl oxidase mRNA levels, lysyl oxidase proenzyme, and its extracellular proteolytic processing, in a dose- and time-dependent manner. Increases in lysyl oxidase mRNA by 400 pM TGF-beta1 were followed to a lesser degree by increases in enzyme activity. Moreover, we found a 6-h delay between the growth factor-stimulated peak in lysyl oxidase steady state mRNA levels and enzyme activity. This suggests that lysyl oxidase biosynthesis is slow, and may be limited by post-translational steps in MC3T3-E1 osteoblastic cells.

Post-translational control of lysyl oxidase was supported by short-term pulse-labeling/immunoprecipitation experiments following TGF-beta1 treatment. Due to the short-term pulse labeling and low level of processing protease(s), the resulting media (extracellular) labeled lysyl oxidase were not processed to the 32-kDa molecular species. Cell-associated radiolabeled lysyl oxidase proenzyme was increased in proportion to TGF-beta-dependent changes in steady state lysyl oxidase mRNA levels, indicating that there was no block of translation of lysyl oxidase pro-protein in TGF-beta1-stimulated cells. Experiments with long periods of continuous pulse labeling resulted in increased accumulation of both 50-kDa precursor and 32-kDa mature enzyme in the media. This supported the concept that lysyl oxidase processing protease(s) partially limited production of the 32-kDa molecular form of lysyl oxidase.

We produced the lysyl oxidase proenzyme as a fusion protein with the maltose-binding protein in E. coli in order to develop an independent assay for lysyl oxidase processing protease(s). Interestingly, the maximum increase in lysyl oxidase enzyme activity (3.4-fold of control) at 15 h was greater than the processing protease activity (1.7-2-fold of control). This finding supported the pulse-labeling studies suggesting that proteolytic processing of lysyl oxidase proenzyme may partially limit the production of the 32-kDa molecular form of lysyl oxidase. Moreover, the inhibition by EDTA and 1,10-phenanthroline, confirmed the notion that this activity is a metalloproteinase(19, 20) .

400 pM TGF-beta1 caused an increase in steady state lysyl oxidase mRNA and in both the 32 kDa ± 3-kDa and 50 ± 5-kDa kDa lysyl oxidase protein molecules, whereas the increase in enzyme activity occurred to a lower extent. These findings may be related to previous studies where a catalytically inactive 32-kDa form of lysyl oxidase was isolated from bovine aorta(33) , and to observations indicating that purified lysyl oxidase is not fully active (34) . Furthermore, TGF-beta1 treatment might not have increased the levels of proteins involved in copper transport and metabolism, which could also have impaired development of full lysyl oxidase enzyme activity(35, 36, 37) . It is also currently unknown whether the 50-kDa molecule secreted by cultured cells is catalytically active, although indirect evidence suggests that this molecular form is inactive. Thus, cell cultures exhibiting lysyl oxidase activity in the cell layer such as neonatal rat aorta smooth muscle cells and rat lung fibroblasts have been shown to accumulate an insoluble 32-kDa form of lysyl oxidase(19, 32) . In contrast, cultures such as the MC3T3-E1 cells grown as described in the present study accumulate no 32-kDa lysyl oxidase in the cell layer, and exhibit no detectable activity in this fraction. All activity was found in the medium. Taken together these studies show that lysyl oxidase activity correlates well with the presence of the 32-kDa molecular form of lysyl oxidase, and not with the 50-kDa molecular form. It is of interest, however, that an active 45-kDa lysyl oxidase has been demonstrated in rat skin tissues(38) .

As MC3T3-E1 cells produce relatively high levels of soluble lysyl oxidase enzyme activity, and 32 kDa- and 50 kDa-immunoreactive proteins, they may provide a source of lysyl oxidase that will allow direct analysis of the activity of these different molecular forms. Elucidation of the mechanisms of activation of lysyl oxidase activity in osteoblasts is required in order to evaluate the ultimate importance of these pathways in bone formation and pathology. It is of interest that lysyl oxidase dependent cross-linking has been shown to be decreased in human osteoporotic bone, and is likely related to the known bone weakness associated with this disease(41) .

MC3T3-E1 cells have been shown to undergo phenotypic changes characteristic of the three proposed stages of osteoblast differentiation, resulting in the production of a mineralized extracellular matrix(3, 39) . We have now shown that TGF-beta1 up-regulated lysyl oxidase activity and mRNA levels consistent with COL1A1 mRNA regulation in preconfluent MC3T3 E1 cells. It will be of interest to further these studies in long-term mineralizing organ and cell cultures, analyzing for phenotype- and growth factor-dependent changes in lysyl oxidase regulation and collagen cross-linking and accumulation. Such studies may reveal minimum and optimum levels of lysyl oxidase activity required for collagenous matrix accumulation. These experiments would further define possible connections between the regulation of lysyl oxidase and TGF-beta1-mediated increases in bone extracellular matrix.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant DE 11004 (to P. C. T.), by a fellowship awarded by the Federal Agency for Post-graduate Education (CAPES) (to E. J. F-F.), Brazil, and by Grants 321/722/94 and 411/722/94 from the Ministry of Education, Finland (to T. E. S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Boston University Goldman School of Graduate Dentistry, Boston University Medical Center, Boston, MA 02118.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor beta; BAPN, beta-aminopropionitrile; COL1A1, alpha-1 type I collagen; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).


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