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Mannose-6-Phosphate/IGF-I1 Receptors Mediate the Effects of IGF-1-Induced Latent Transforming Growth

Factor fl1 on Expression of Type I Collagen and Collagenase in Dermal Fibroblasts

AZIZ GHAHARYa,*, EDWARD E. TREDGETa, QIONG SHENa, RUHANGIZ T. KILANI", PAUL G. SCOTTb and YVONNE HOULEa

'Department of Surgery, Wound Healing Research Group. bDepartment of Biochemistry, University of Alberta, Edmonton. Alberta, Canada. T6G 2B7

(Received 19 Februar-v 1999; Revised 21 May 1999; In finalform 26 July 1999)

We have previously shown that insulin-like growth factor-1 (IGF-1) induces the expression of latent transforming growth factor B1 (LTGF-PI) through activation of c-fos and c-jun oncogenes. In this study we investigated whether IGF-1 induced latent TGF-Bl has autocrine effects on dermal fibroblasts and described a possible mechanism. Human dermal fibroblasts were treated with either vehicle, IGF-1 alone, or IGF-1 with either anti-TGF-Bl neutralizing antibody or mannose-&phosphate (M6P) and levels of mRNAs for TGF-B1, collagenase and the pro al(1) chain of type I collagen were then evaluated by Northern analysis. Conditioned medium was also collected from treated and untreated cells and assayed for TGF-B1 protein by enzyme-linked immunosorbent assay. The results of the Northern analysis revealed a differential effect on the expression of the pro al(1) chain of type I collagen and collagenase in dermal fibroblasts: mRNA for the former being significantly increased in response to IGF-1 treatment while that for collagenase was markedly suppressed. These effects of IGF-1 were blocked to a significant extent by TGF-B1 neutralizing antibody a t a concentration of 0.5- 2.0 pg/ml. As the TGF-Bl induced by IGF-1 is inactive in the traditionally used mink lung epithelial cell growth inhibition assay, we explored the possible role of IGF-II/M6P receptors in facilitating these autocrine effects. The results showed that the greater than two-fold in- crease (201.9 k 38 vs 81.8 f 13,p < 0.05) in mRNA for the pro al(1) chain of type I collagen induced by IGF-1 was at least 60% inhibited by M6P in a time-dependent fashion. A direct correlation between the expression of TGF-B1 and the pro al(1) chain of type I collagen was found in response to either IGF-1 alone or IGF-1 with M6P. Treatment of cell cultures with TGF-B1 neutralizing antibody mimicked the effect of M6P. In contrast to the effects on expression of type I collagen, the level of collagenase mRNA was markedly reduced by IGF-1 alone and was restored by the administration of M6P. The levels of TGF-B1 in conditioned

* Corresponding author. 161 HMRC, 8440-1 12 Street, University of Alberta, Edmonton, Canada, T6G 2B7. Tel.: (780)492-8603. Fax: (780)492-6361.

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168 A. GHAHARY et a / .

medium from treated and untreated cells showed a similar pattern to that of the mRNA detected by Northern analysis. These findings suggest that IGF-1 induces latent TGF-Bl and that the matrix-modulating autocrine effects of LTGF-Bl on dermal fibroblasts are facilitated by M6P/IGF-I1 receptors on these cells.

Keywords: IGF- I , TGF-01, mannose-6-phosphate, collagen dermal fibroblasts

Abbreviations: mRNA, Messenger ribonucleic acid; ECM, Extracellular matrix; FBS, Fetal bovine serum; PBS, Phosphate buffered saline; kb, Kilobase; IGF-1 and 11, Insulin-like growth factor- 1 and 11; TGF-01, Transforming growth factor-pl ; FGF, Fibroblast growth factor; PDGF, Platelet- derived growth factor; LAP, Latency associated peptide; LTBP, Latent TGF-0 binding protein; AP-1, Activating protein 1; M6P, Mannose-6-phosphate

INTRODUCTION

The efficacy of a cytokine or growth factor in physiological and pathological conditions depends on its interaction with other growth factors present in the tissue environment. IGF-1 and TGF-P1 are two multi-functional factors that are co-expressed by many different cell types such as fibroblasts, platelets and activated macrophages. In many tissues, fibroblasts, which are the primary source of ECM, synthesize both TGF-P1 and IGF-1 and respond to these growth factors (Ghahary et al., 1994; Barreca et al., 1992). Chondrocytes are another cell type which are able to express both growth factors and respond to them (Hill et al., 1992). During tissue repair, activated platelets (Karey and Sirbasku, 1989; Sporn et al., 1987) and macrophages (Sporn et al., 1987; Nagaoka et al., 1990) release IGF-1 and TGF-PI at the site of injury and these are detectable in local tissues and in wound chambers (Spencer et al., 1988; Grotendorst c’t al., 1988).

There is now compelling evidence that both IGF-I and TGF-P1 function as fibrogenic factors. IGF- 1 stimulates collagen production by human lung fibroblasts (Goldstein et al., 1989), bovine fetal growth-plate chondrocytes (Hill et al., 1992) and human dermal fibroblasts (Bird and Tyler, 1994). Similarly, TGF-P1 also stimulates collagen produc- tion in normal human dermal fibroblasts (Varga et a/., 1987). Both growth factors have been shown

to stimulate DNA synthesis, replication of isolated epiphyseal growth-plate chondrocytes, induction of glycosaminoglycans and incorporation of radio- labeled proline into collagen in these cells (Hill et al., 1992; O’Keefe et al., 1988; Makower et al., 1989). Despite the fact that these growth factors are present in many tissues, are co-expressed by many different cell types and function as fibrogenic factors, their interaction has not previously been examined. We have recently provided evidence (Ghahary et al., 1998) that IGF-1 stimulates the expression of TGF- P l mRNA in a dose-dependent fashion through an increase in the rate of transcription. The mink lung epithelial cell growth inhibition assay revealed that the TGF-/3I released into conditioned medium of IGF- 1 treated cells was biologically inactive. This was not surprising as it is clear from the literature that TGF-Pl is secreted as a latent high molecular weight complex (LTGF-P), consisting of a 25 kDa dimeric mature protein, with 112 amino acids in each subunit, and the dimeric N-terminal precursor peptide known as latency associated peptide (LAP) (Tsuji et al., 1990). As released from fibroblasts, platelets, and bone cells, the latent TGF-PI complex also contains a protein termed the latent TGF-/3 binding protein (LTBP) which is a 125- 205 kDa glycoprotein covalently associated with the LAP. Unlike LTBP expressed by fibroblasts and bone cells, LTBP in platelets is a truncated complex. Latent TGF-P1 produced by some osteo- blast-like cells, growth plate chondrocytes, normal

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MANNOSE-6-PHOSPHATE SUPPRESSES IGF- 1 -INDUCED TGF-01 169

calvarial bone cells and recombinant TGF-PI synthesized by Chinese hamster ovary cells, is not associated with LTBP (Dallas et al., 1994; Moren et al., 1994; Pedrozo et al., 1998; Bonewald et al., 1991).

Most cell types possess TGF-PI receptors and activation of LTGF-P1 may be a critical regulatory event in vivo (Sporn et al., 1986). Although transient acidification, proteolysis and chaotropic agents can all activate LTGF-/3 in vitro (Lyons et al., 1988), it is not known for certain how this occurs in vivo. Several different possible mechanisms have recently been reported. Schultz-Cherry et al. (1 994) provided evidence that the chymotrypsin-resistant core of thrombospondin- 1 binds and activates the latent TGF-P1 derived from aortic endothelial cells in culture. Kovacina et al. (1989) reported that 1251- labeled LTGF-/3 binds to the cell membrane and suggested that exposure of internalized LTGF-P and receptors to the acidic conditions of the endo- somal/lysosomal compartments could then activate it. A role of LTBP in the activation of LTGF-P has also been reported. Flaumenhaft et al. (1993) demonstrated that activation of LTGF-P normally seen when endothelial cells are co-cultured with smooth muscle cells can be inhibited by addition of antibodies to native platelet LTBP. These investi- gators suggested that LTBP concentrates LTGF-/3 on the cell surface where activation occurs. Al- though none of these mechanisms for activation of latent TGF-P1 has been proven to operate in vivo, they are potentially important in physiological conditions. In the present study we hypothesize that latent TGF-Pl induced by IGF-1 has autocrine effects on dermal fibroblasts through interaction with the non-signaling insulin like growth factor II/ mannose-6-phosphate (IGF-II/M6P) receptors on these cells. The hypothesis is based on the fact that Dennis and Rifkin (1991) found that the LTGF-P complex binds to human IGF-II/M6P receptors in vitro. The expression of the pro al(I) chain of type I collagen and collagenase, as two key ECM components produced primarily by fibroblasts, was chosen as an index for autocrine effects of latent TGF-Pl induced by IGF-1. This study provides

evidence that IGF-II/M6P receptors facilitate the autocrine effects of latent TGF-P1 induced by IGF-1 in dermal fibroblasts.

MATERIALS AND METHODS

Cell Cultures and Treatment

Patients who were treated at the University of Alberta Hospital Firefighters’ Burn Treatment Unit were subjects for this study. Following informed consent, normal skin punch biopsies were obtained from patients undergoing elective reconstructive surgery. Cultures of fibroblasts were established as previously described (Ghahary et al., 1995). Strains of dermal fibroblasts at passages 3-7 were used in this study. To establish the optimum time at which IGF-I induces mRNA for TGF-Pl and type I collagen, in a pilot study dermal fibroblasts were treated with IGF-1 (Genentech Inc., San Francisco, CA, USA) at a final concentration of 100 ng/ml for a duration of either 0, 6, 12, or 24 h. Twenty four hours treatment with IGF-1 was found to be opti- mal and was used for subsequent experiments. As fetal bovine serum (FBS) contains relatively high levels of TGF-P1 and IGF-1, medium with a low concentration (0.2%) of FBS was used in experi- ments to evaluate TGF-PI by enzyme-linked im- munosorbent assay (ELISA) and the mink lung epithelial cell proliferation assay. The effects of IGF-1 induced latent TGF-P1 were evaluated by treating the fibroblasts either with vehicle, IGF-1 alone or IGF-1 with various concentrations (0.25, 0.5, 1.0, and 2.0 lg/ml) of anti-TGF-pl neutralizing antibodies (R&D Systems) in DMEM supplemen- ted with 2% FBS for 24 h. Total RNA from treated and untreated cells was then extracted and evaluated by Northern analysis. In another experiment, cells were treated with either vehicle, IGF-1 alone or IGF-1 with 1 mM of M6P and the expression of mRNAs for the pro al(I) chain of type collagen, collagenase and TGF-Pl were evaluated by North- ern analysis. To determine the time frame for M6P to inhibit the effects of IGF-1, dermal fibroblasts

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170 A. GHAHARY et a/.

were treated with either vehicle, IGF- 1 alone for 48 h or IGF- 1 and 1 .O mM M6P for 12,24,36 or 48 h.

Enzyme-Linked Immunosorbent Assay for TGF-Bl

A sandwich ELISA based on the procedure re- ported by Danielpour et al. (1989) was used. Briefly, 96-well plates were coated with loop1 per well of monoclonal antibody to human TGF-P1, 2, and 3 (R&D Systems) at a concentration of 1 pg/ml in PBS. The plates were incubated for 2 h at room temperature (RT) followed by 16 h at 4°C. After washing twice with PBS-Tween-20 (PBS-T), the plates were blocked with 1 YO bovine serum albumin (BSA, crystallized, Sigma) for 60min at RT and washed three times with PBS-T. The culture media were acidified with 24pl per ml of 5 N HCI for 15 min at RT and neutralized with 40 pl per ml of 1 M HEPESj5 N NaOH (5/2). One hundred micro- litres of each acidified/neutralized sample was added to each well on the plates, which were then incubated at RT for 60min. After washing, the plates were incubated with 100 p1 per well ofchicken anti-human TGF-y (R&D Systems), which recog- nizes all three mammalian TGF-/3 isoforms, at a concentration of 2.5 pg/ml for 60min at RT with shaking. After washing five times with PBS-T, the plates were incubated with alkaline phosphatase- conjugated rabbit anti-chicken IgG (Sigma) at RT for 60min followed by washing five times with PBS-T. After addition of the substrate (o-nitro- phenyl phosphate, 1 mgjml, Sigma), the plates were incubated at RT for 60 min and the optical density was read using a THERMOmaxTM (Molecular Devices, Menlo Park, CA) microplate reader at a wavelength of 405 nm. Serial dilutions (0, 125, 250, 625, 1250, to 2500pg/ml) of recombinant human TGF-P1 (Genzyme) were used to prepare a standard curve.

Extraction of RNA and Northern Analysis

Following treatment, culture medium was removed and cell layers were lysed in 6ml of

guanidinium thiocyanate (GITC) as previously described (Ghahary et al., 1995). Total RNA was extracted by the GITC/CsCI procedure of Chirg- win et al. (1979), separated by electrophoresis and blotted onto nitrocellulose filters. Filters were then baked under vacuum for 2 h at 80°C and pre- hybridized in a solution containing 50% forma- mide, 0.3 M sodium chloride, 20 mM Tris-HC1 (pH KO), 1 mM EDTA, 1 x Denhardt’s solution (1 x = 0.02% BSA, Ficoll and polyvinylpyrroli- done), 0.005% salmon sperm DNA, 0.005% poly(A) for 2-4h at 45°C. Hybridization was performed in the same solution at 45°C for 16- 20 h using cDNA probes for human TGF-PI, pro cyl(1) of type I collagen or collagenase. The TGF- ,6l cDNA probe was an EcoR-1-EcoR-1 fragment bearing the full length TGF-P1 coding region. Similarly, the collagen pro al(1) chain probe was an EcoR- 1 -EcoR- I DNA fragment of approxi- mately 4.0 kb specific for human type I collagen. The blots were subsequently re-hybridized with cDNA specific for 18s ribosomal RNA as a control for loading. The probes were labeled with 32P-a-dCTP (DuPont Canada, Streetsville, Mississauga, Ontario, Canada) by nick-transla- tion. Filters were initially washed at RT with 2x SSC (1 x = 0.15 M sodium chloride, 0.01 5 M sodium citrate) and 0.1% SDS for 30min, then for 20min at 65°C in 0 . 1 ~ SSC and 0.1% SDS solution. Autoradiography was performed by exposing Kodak X-Omat film to the nitrocellulose filters at -70°C in the presence of an enhancing screen. The cDNA probes for the pro al(1) chain of type I collagen and TGF-P1 were kindly pro- vided to us by Drs. G. Tromp H. Kuivaniemi, and L. Ala-Kokko (Department of Biochemistry and Molecular Biology, Jefferson Institute of Molec- ular Medicine, Philadelphia, PA, USA) and Dr. G.I. Bell (Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biol- ogy and Medicine, University of Chicago, IL, USA), respectively. The cDNA probes for collage- nase and 18s ribosomal RNA were obtained from American Type Culture Collection (12301- Parklawn Drive, Rockville, MD, USA).

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MANNOSE-6-PHOSPHATE SUPPRESSES IGF- 1-INDUCED TGF-Bl 171

Statistical Analysis

The statistical significance of differences in the quantity of TGF-01 protein and the mRNA for pro cwl(1) of type I collagen were tested using Student’s t-test. P values < 0.05 were considered significant.

RESULTS

Induction of TGF-B1 mRNA and Protein in Response to IGF-1 and IGF-1 Plus M6P

We have previously demonstrated that TGF- 1 induces TGF-Pl mRNA expression in a dose- dependent fashion. Here, we demonstrate that IGF-1 stimulates the expression of TGF-PI which in turn induces mRNA for the pro cyl(1) chain of type I collagen in a time-dependent manner (Fig. 1). As shown in this figure, a cDNA probe specific for human TGF-PI visualized a transcript with appar- ent size of 2.5 kb whose level increased markedly in IGF-1 treated fibroblast cells as early as 6 h

IGF-1 Treatment 0 6 12 24h

TGF-B1

28s

FIGURE 1 Effect of time of treatment with IGF-I on ex- pression of mRNA for TGF-01 and type I procollagen. Der- mal fibroblasts were treated with 100ng/ml of IGF-I for 0, 6, 12, or 24h and total RNA was extracted, separated by elec- trophoresis on a 1% agarose gel and blotted onto nitrocellu- lose. The blots were initially hybridized with cDNAs for TGF-01 and subsequently for the pro al(1) chain of type I procollagen. The intensity of ethidium bromide staining for 28s rRNA was used as a control for loading. The autoradio- gram shown here is representative of two blots obtained from two different experiments.

and reached its maximum level within 24 h. The expression of collagen pro cwl(1) chain transcripts with apparent sizes of 4.8 and 5.8 kb was increased by 12 h. This finding indicates that an increase in IGF-1 induced TGF-P1 precedes the increase in expression of type I collagen mRNA.

To examine whether IGF-1 -induced latent TGF- P1 was functional, Northern analysis was used to evaluate the expression of TGF-PI, type I collagen and collagenase mRNA by dermal fibroblasts in response to either vehicle, IGF-1 or IGF-1 plus M6P. As shown in Fig. 2, IGF-1 alone markedly increased the expression of the TGF-Pl transcript relative to untreated cells and this induction was suppressed by M6P. To confirm that this increase in mRNA for TGF-Pl was accompanied by an

C IGF IGF

M6P +

18s

FIGURE 2 Expression of mRNA for TGF-PI, the pro cul(1) chain of type I collagen and collagenase in response to IGF-I and IGF-l plus M6P. Dermal fibroblasts were treated with either vehicle (C), IGF-1 alone or IGF-l+M6P for 48h as described in the Materials and Methods. Procedure for RNA extraction and Northern analysis was the same as in Fig. I . The blots were initially hybridized with cDNAs for TGF-PI and subsequently for the pro cul(1) chain of type I procolla- gen, collagenase and 18s rRNA. Except for the pro al(1) chain of type I collagen which is a representative of the four differ- ent experiments, the autoradiogram shown here is representa- tive of two blots obtained from two different experiments.

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x-

T

C IGF-1 IGF-l+M6P TREATMENT

FIGURE 3 Quantitative analysis of TGF-PI induction in response to IGF-1 alone and IGF-I plus M6P. Conditioned medium from either vehicle (C) , IGF-1 alone or IGF-l+M6P was collected and the TGF-PI was measured by ELISA. Data are expressed as mean f SEM for four separate experiments done in triplicate. The asterisk (") denotes a significant differ- ence in TGF-Dl induction between either control and IGF-1 or IGF-1 and IGF-I+M6P treated cells.

increase in protein, the levels of TGF-P1 in condi- tioned medium were evaluated by ELISA. As shown in Fig. 3, there was a significant increase (10.5 f 1.3 vs 6.1 * 0.8 pg/104 cells, n = 4, p < 0.05) in the concentration of this cytokine in conditioned medium obtained from IGF- 1 -treated cells. Addi- tion of 1 mM M6P suppressed the induction of TGF-PI by IGF-I (6.7 * 1.6 vs 10.5 f 1.3 pg/104 cells, n = 4, p < 0.05).

Inhibition of the IGF-1 Induced Modulation of Type I Collagen and Collagenase mRNA Expression by Mannose-6-Phosphate

As shown in Fig. 2, the blot previously hybridized with TGF-01 cDNA was re-hybridized withcDNAs specific for the pro a1 chain of type I collagen or collagenase. The result demonstrated that the expression of the 4.8 and 5.8 kb transcripts for this protein responded to IGF-1 alone and IGF-I plus M6P similarly to that of TGF-PI. In contrast, the

x-

T

c IGF-1 IGF-liM6P TREATMENT

FIGURE 4 Quantitative analysis of mRNA for the pro crl(1) of type I collagen in response to IGF-1 alone and IGF- 1 plus M6P. Four separate autoradiograms similar to that shown in Fig. 2 were quantified by densitometery and data are expressed as mean f SEM in densitometry units. The as- terisk (3 denotes a significant difference between results for control and IGF-1 or between IGF-1 and IGF-1 plus M6P treated cells.

expression of collagenase mRNA was markedly suppressed by IGF-I and restored with M6P. This indicates a differential effect of IGF-I on expres- sion of these ECM proteins. Three more strains of dermal fibroblasts were treated under the same conditions and expression of mRNA for the pro cyI(1) of type I collagen was evaluated by Northern analysis. Quantitative analysis of these autoradio- grams revealed a greater than two-fold increase (201.93~ 38vs81.8 f 13densitometryunits,p < 0.05, n = 4 ) in the steady state level of this mRNA in response to IGF- 1 treatment and, as shown in Fig. 4, more than 70% of this increase was suppressed by M6P(113.8f27vs201.9f38, densitometry units, p < 0.05, n = 4).

Time Dependence of the Inhibition by Mannose-&Phosphate of Type I Collagen mRNA Induction by IGF-1

The efficacy of M6P inhibition of IGF-1 induction of the pro a 1 (I) chain of type I collagen was explored

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MANNOSE-6-PHOSPHATE SUPPRESSES IGF-I-INDUCED TGF-Dl 173

M6P Added for 48 36 24 12 - - h IGF-I + + + + + -

18 s

FIGURE 5 Time dependency of effects of M6P on expres- sion of mRNA for the pro al(1) chain of type I collagen. Dermal fibroblasts were treated with either vehicle, IGF-1 alone for 48h or IGF-I for 48h plus M6P for either 12, 24, 36, and 48 h. The blots were initially hybridized with cDNAs for the pro al(1) chain of type I procollagen and subsequently with cDNA specific for 18s rRNA as a control for loading. The autoradiogram shown here is representative of the two separate blots obtained from two different experiments. Other details as for Fig. 1.

as a function of time. The result of this experiment shown in Fig. 5 , revealed that this effect was maximally abrogated by M6P added for 36-48 h. The efficacy of M6P diminished as the time of exposure to M6P was reduced. This finding in- dicates that the inhibitory effect of M6P is time dependent.

Effect of Anti-TGF-Bl Neutralizing Antibody on Type I Collagen mRNA Induction by IGF-1

Since TGF-P1 can stimulate its own production (Kim et al., 1990), we also considered the question of whether part of the increase in TGF-P1 in response to IGF-1 treatment resulted from such autoinduc- tion. We have previously demonstrated that cells treated with IGF-1 in the presence of anti-TGF-Pl neutralizing antibody showed more than 50% reduction in induced TGF-P1 (Ghahary et al., 1998). To confirm the specificity of the mediator effects of TGF-01 in induction of type I collagen by dermal fibroblasts, we evaluated the expression of mRNA in the presence of various concentrations (0, 0.25, 0.5, 1 .O, and 2.0 pg/ml) of anti-TGF-P1 neutralizing antibody. The results shown on Fig. 6 revealed that higher concentrations (0.5-2 pg/ml)

TGF-f31 Antibody - - 0.25 0.5 1.0 2.0 pg - + + + + + IGF-1 -

Pro al(I) - Collagenase -

18 S

FIGURE 6 Dose dependency of effects of anti-TGF-01 neu- tralizing antibody on expression of mRNA for the pro al(I) chain of type I collagen. Dermal fibroblasts were treated with either vehicle, IGF-1 alone or IGF-I plus various concentra- tion (0.25, 0.5, 1.0, and 2.0pg/ml) of anti-TGF-pl neutraliz- ing antibody for 48 h. Other details as for Figs. 1 and 4.

of this antibody markedly suppressed the effect of IGF- 1.

DISCUSSION

In our previous study (Ghahary et al., 1998), we demonstrated that IGF- 1 induced TGF-PI through a transient expression of mRNA for c-fos and c-jun, two components of the AP-1 complex known to be involved in the autoinduction of TGF-P1 . However, the induced TGF-P1 was latent as tested by the traditionally used mink lung cell growth inhibition assay. In this study, we explored whether latent TGF-01 induced by IGE-1 is functional for dermal fibroblasts and, if so, what mechanism of activation might be involved. As Dennis and Rifkin (1991) reported that the LTGF-P complex binds to human M6P receptors in vitro, we hypothesized that latent TGF-P1 may interact with IGF-II/M6P receptors on dermal fibroblasts and that this could facilitate the matrix-modulating effects. The results clearly show that induction of latent TGF-PI in response to IGF-I is inhibited by M6P. This inhibition is pro- bably specific to M6P because the previous report by Dennis and Rifkin (1991) demonstrated that neither glucose-6-phosphate nor mannose- 1 -phosphate

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inhibited interaction of LTGF-/31 with IGF-II/M6P receptors. Further, the results of our own recent study (Ghahary et al., in press) showed only M6P, but not G6P, inhibits the effects of LTGF-01 released by genetically modified keratinocytes co- cultured with dermal fibroblasts. This finding suggests that IGF-II/M6P receptors are involved either directly or indirectly in IGF-1 induced latent TGF-PI autoinduction. Latent TGF-01 has three glycosylation sites on Asn-82, Asn-136, and Asn- 176. Two of these three glycosylation sites are phosphorylated (Miyazono and Heldin, 1989). Studies with IGF-II/M6P receptors have shown that the TGF-01 precursor binds and that the binding is specifically inhibited by M6P (Kovacina et a/., 1989). There are two specific cell membrane receptors for M6P, one of which is cation-indepen- dent (270 kDa) and is structurally identical to the IGF-I1 receptor (Morgan et al., 1987). Thus, one possibility is that an excess (1 mM) of M6Pcompetes with M6P groups on latent TGF-PI for IGF-III M6P receptors on dermal fibroblasts and thereby suppresses its effects.

The second objective was to examine whether latent TGF-/31 induced by IGF-1 had any matrix- modulating effects on dermal fibroblasts. To ad- dress this issue, one of the major components of extracellular matrix, type I collagen, primarily ex- pressed by dermal fibroblasts, was studied. TGF-Pl strongly stimulates expression of type I collagen by dermal fibroblasts (Varga et al., 1987). The results revealed that IGF- 1 stimulates the expression of TGF-/31 mRNA at least 6 h earlier than that for type I collagen. An early expression of IGF-1 induced TGF-/31 is not surprising as we have pre- viously demonstrated that IGF- 1 markedly in- creased the expression of c-fos and c-jun, the two proteins of the AP complex, within 30-60min of IGF-1 treatment (Ghahary et al., 1998). These findings indicate that the induction of type I col- lagen is likely to be mediated through IGF-1 induced TGF-/31 expression. Addition of M6P mar- kedly reduced the effects of IGF-1 on expression of pro al(1) of type 1 collagen mRNA. The specificity of this effect was demonstrated by re-hybridization

of the same blot with cDNA specific of collagenase showing a differential effect of IGF-1 alone and IGF-1 plus M6P on expression of collagenase mRNA relative to that of type I collagen.

Data obtained from the study of the time- dependent M6P inhibition clearly demonstrated that this agent was maximally effective when cells were treated 36-48 h. This inhibitory effect was less pronounced when M6P was present in culture medium for a shorter period. This suggests that induction of type I collagen mRNA expression in response to IGF-1 is secondary and may be mediated through IGF- 1 -induced latent TGF-P1 . This is also supported by the results obtained from administration of anti-TGF-P1 neutralizing anti- bodies in a similar experimental setting as M6P. As shown for M6P, IGF-I alone and IGF-1 plus various concentrations of antibody had a differen- tial influence on expression of mRNA for collage- nase relative to that of the pro aI(1) chain of type I collagen. This confirmed our previous results that anti-TGF-P1 neutralizing antibody significantly suppressed the effect of IGF- 1 on TGF-01 produc- tion by dermal fibroblasts (Ghahary et al., 1998). This finding also suggests that not all TGF-PI present in conditioned medium of IGF- 1 -treated cells results from a direct effect of IGF-1 but rather that autoinduction is at least partly responsible. Hill et a/ . (1992) recently reported a greater efficacy of IGF-1 in stimulation of radiolabeled proline in- corporation into collagen by chondrocytes relative to TGF-PI. As both IGF-1 and TGF-Pl have been reported to stimulate collagen production (Hill et al., 1992; O’Keeje et a/., 1988; Makower et al., 1989), the greater effect of IGF- 1 might be due to the combination of direct and indirect (IGF-1 induced TGF-/31 autoinduction) activity.

The inhibitory effect of M6P on induction of type I collagen in response to IGF-1 substantiates the hypothesis that the mechanism for activation of LTGF-PI is dependent on IGF-II/M6P receptors, although a role for cell-membrane associated pro- teinases is not excluded by our study. Further sup- port for the importance of M6P receptors comes from the observation that these seem to be required

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MANNOSE-6-PHOSPHATE SUPPRESSES IGF-1-INDUCED TGF-Dl 175

for activation of LTGF-P in co-cultures of bovine aortic smooth muscle and endothelial cells. In this system, the two cell types must be either in contact or in close proximity (Dennis and Rifkin, 1991). De Bleser et al. (1995) recently demonstrated that IGF-II/M6P receptors on fat-storing cells are ne- cessary but not sufficient for the activation of LTGF-P1 in carbon tetrachloride induced fibrotic rat liver.

It is not clear how interaction of latent TGF-,!?l with IGF-II/M6P receptors results in activation of the TGF-,!? receptors. As proposed by Kovacina et al. (1989), one possibility is that the receptor- ligand complex is internalized and exposed to the acidic conditions of the endosomal/lysosomal com- partments which activates the LTGF-P1. Other possibilities are that the interaction of LTGF-PI with the IGF-II/M6P receptor brings it into contact with cell membrane-associated proteinases which cleave off the LAP or that this interaction causes a change in conformation allowing the binding site of TGF-P1 to interact with the receptors. Although matrix effects and autoinduction of TGF-PI ex- pression due to exogenous administration of re- combinant TGF-PI are well-documented (Kim et al., 1990), the role of latent TGF-,!?I as an auto- crine factor in vitro and in vivo has not been elu- cidated because TGF-P1 is reportedly inactive due to its association with the latency associated protein (Tsuji et al., 1990). Thus, our findings could be the first indication that latent TGF-PI has, at least in our system, functional significance. The mechan- isms involved are currently under investigation.

In conclusion, data presented here provide evi- dence that IGF-l-induced latent TGF-PI is func- tional for dermal fibroblasts and that its effects are mediated through interaction with ICF-II/M6P receptors.

Acknowledgments

This research was supported by the Medical Re- search Council of Canada (A. Ghahary ) and the University of Alberta Firefighters’ Burn Trust Fund. The IGF-1 used in this study was kindly

provided by Genentech Inc. San Francisco, CA, USA.

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