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Cell Growth & Differentiation Vol. 10, 721-728, November 1999
© 1999 American Association for Cancer Research

Articles

Loss of Insulin-like Growth Factor I Receptor-dependent Expression of p107 and Cyclin A in Cells That Lack the Extracellular Matrix Protein Secreted Protein Acidic and Rich in Cysteine1

Amitabha Basu, Ulrich Rodeck, George C. Prendergast and Chin C. Howe2

The Wistar Institute, Philadelphia, Pennsylvania 19104 [A. B., G. C. P., C. C. H.], and Department of Dermatology and Cutaneous Biology, Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [U. R.]

Abstract

The extracellular matrix-associated glycoprotein secreted protein acidic and rich in cysteine (SPARC) has been implicated in the control of cell proliferation during tissue remodeling, wound healing, and malignant development. Here, we describe a novel mechanism through which SPARC influences cell cycle progression in embryonic fibroblasts derived from Sparc-nullizygous (-/-) mice. SPARC-deficient cells were indistinguishable from wild-type cells in their ability to initiate DNA synthesis after treatment with either fetal bovine serum or platelet-derived growth factor. In contrast, Sparc -/- cells responded poorly to activation of the insulin-like growth factor receptor (IGFI-R) by insulin. This defect was traced to reduced expression of the IGFI-R in Sparc -/- cells. Consistent with impaired cell cycle progression through S-phase, insulin-stimulated Sparc -/- cells also revealed reduced expression of two key regulators of S phase progression (cyclin A and thymidine kinase), whereas expression of the G1 phase progression regulators c-myc or cyclin D1 was unaffected. An examination of the status of retinoblastoma family pocket proteins in Sparc -/- cells revealed a selective and dramatic reduction in levels of the retinoblastoma-related protein p107. Exogenous platelet-derived growth factor restored expression of the IGFI-R and IGFI-R dependent DNA synthesis as well as induction of cyclin A, thymidine kinase, and p107 in insulin-stimulated Sparc -/- cells. These results suggest that SPARC-dependent matrix to cell interactions contribute to the regulation of p107 and cyclin A through IGFI-R dependent pathway(s).

Introduction

SPARC,3 also known as osteonectin, BM-40, or 43K protein (1, 2, 3) , is an ECM-associated glycoprotein believed to interact with several ECM molecules, such as collagens and vitronectin, and cell surface receptors (4, 5, 6) . SPARC is widely expressed in many cell types and different tissues in both embryonic and adult mice, often at high levels (7, 8, 9) . Among various cell types, fibroblasts and endothelial cells express high levels of SPARC (5 , 6) . SPARC is also strongly expressed in rapidly growing tissues in the embryo and during wound healing in the adult (10 , 11) . Many invasive cancers exhibit elevated expression of SPARC, including melanomas and tumors of the gastrointestinal tract, breast, lung, kidney, adrenal cortex, and brain (10 , 12) . Antisense strategies that inhibit SPARC expression in melanoma cells cause a reduction in ECM adhesion and invasive potential, implying a role for SPARC in tumor progression (13) . However, putative roles for SPARC in support of tumor progression are cell type specific, because overexpression of SPARC in ovarian cancer cells has been shown to reduce tumorigenic potential (14) .

SPARC exerts complex effects on cell shape, adhesion, and proliferation. For example, in primary fibroblasts or endothelial cells, the addition of exogenous SPARC induces cell rounding and disengagement from the ECM in a Ca2+-dependent manner (15 , 16) . In endothelial cells, exogenous SPARC also inhibits proliferation (17 , 18) . In contrast, in the absence of Ca2+, SPARC facilitates matrix attachment and spreading of fibroblasts. These results suggest that exogenous SPARC can exert diverse effects on cell-matrix interactions and proliferation, depending on cell type and environmental conditions. A clear definition of the function of this protein has been complicated by the fact that most cell types already produce endogenous SPARC that can potentially obscure or modify the effects of exogenously added SPARC.

We addressed the role of SPARC in cell cycle progression by analyzing embryonic fibroblasts derived from SPARC-null mice. We describe that SPARC-deficient cells initiate DNA synthesis normally in response to the competence factor, PDGF, but respond poorly to the progression factor, IGFI. This defect was accompanied by a reduction in expression of the IGFI-R. Furthermore, expression of three key regulators of S phase progression, i.e., p107, cyclin A, and tk, were similarly reduced in SPARC-null cells. The addition of PDGF along with insulin restored wild-type levels of the IGFI-R, p107, cyclin A, and tk. Our findings imply that SPARC-dependent signals control the expression of p107 and cyclin A via an IGFI-R-dependent pathway.

Results

Defective Cell Cycle Progression in SPARC-deficient Cells.
Primary fibroblasts were prepared from 13-day embryos from either wild-type mice or mice bearing a targeted disruption of the Sparc gene (9) . Previous studies have shown that, depending on culture conditions, exogenous SPARC regulates adhesion of fibroblasts to tissue culture dishes (16 , 19) . However, we observed that cultures of Sparc -/- cells seeded on tissue culture-treated plastic dishes attached and spread in a manner that was indistinguishable from wild-type fibroblasts (data not shown). Exogenously added SPARC has been shown to modulate proliferation of wild-type fibroblasts (16) . We, therefore, compared the ability of serum-starved SPARC -/- and wild-type fibroblasts to progress through S phase when treated with FBS or defined growth factors. Because PDGF and IGFI are the essential competence and progression factors in serum responsible for inducing G0-S transit in fibroblasts (20 , 21) , we focused on the response to these factors. Insulin was used at supraphysiological concentrations (2 mg/ml) to activate the IGFI-R as described earlier (22) .

We observed that SPARC -/- fibroblasts incorporated [3H]thymidine as efficiently as control fibroblasts when treated with either FBS or PDGF (Fig. 1A)Citation . In contrast, Sparc -/- fibroblasts incorporated {approx}60% less [3H]thymidine than wild-type cells when treated with insulin (Fig. 1A)Citation . Consistent with an earlier report that exogenous SPARC does not integrate properly into the ECM (23) , the addition of SPARC did not relieve the deficit in DNA synthesis seen in insulin-treated SPARC -/- cells. By contrast, PDGF restored wild-type levels of DNA synthesis when added together with insulin, indicating that the reduced response of SPARC -/- cells to insulin was not attributable to induction of a dominant-acting cell cycle inhibitor. Next, we addressed the question of whether the reduced DNA synthesis in Sparc -/- cells in response to insulin was attributable to fewer cells entering S phase within the time frame of the experiment (10–40 h) or simply to a delay in progression to S phase. To this end, we determined the kinetics of [3H]thymidine uptake over time by pulse labeling cells at various time points after stimulation with growth factors (Fig. 1B)Citation . When treated with insulin, wild-type and SPARC -/- fibroblasts commenced DNA synthesis within 15–20 h, but SPARC -/- cells incorporated overall less [3H]thymidine than wild-type fibroblasts. This result was confirmed by fluorescence-activated cell sorter-assisted cell cycle analysis after staining with propidium iodide. Specifically, 20 h after treatment with insulin, 26.1% of wild-type cells were in the S-G2-M phases of the cell cycle as compared with 16% of SPARC -/- cells. Taken together, these results indicated that the difference in insulin-dependent DNA synthesis between wild-type and SPARC -/- cells was attributable to fewer cells responding to this growth factor.



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  		Fig. 1. DNA synthesis of serum-deprived SPARC-deficient embryonic fibroblasts in response to defined growth factors and FBS. A, results of continuous labeling experiment to determine [3H]thymidine uptake of SPARC -/- ({diamondsuit}) and wild-type fibroblasts ({square}) at various time points after restimulation with insulin, PDGF, insulin plus PDGF, insulin plus SPARC, and serum as indicated. [3H]Thymidine was added together with growth factors throughout the duration of the experiment. [3H]Thymidine incorporation is expressed relative to the insulin-induced incorporation in wild-type cells at 20 h scaled as 100%. B, results of a pulse-labeling experiment to determine time-dependent changes in [3H]thymidine incorporation in SPARC -/- ({diamondsuit}) and wild-type fibroblasts ({square}) upon restimulation with insulin alone, insulin and PDGF, or serum. At each time point, cells were pulse-labeled with [3H]thymidine for 1 h prior to harvesting. Data were derived from experiments performed in triplicate with cells derived from different embryos; bars, SD.

 
Reduced Expression of the IGFI-R in SPARC-deficient Cells.
These results suggested that SPARC -/- fibroblasts had a defect specific to insulin or IGFI-dependent signaling pathways that could be compensated for by PDGF treatment. PDGF has been shown by others to up-regulate expression of the IGFI-R (24 , 25) . On the basis of these findings, we speculated that Sparc -/- cells express comparatively low levels of the IGFI-R, which could be up-regulated by PDGF. Western blot analysis of IGFI-R expression in SPARC -/- and wild-type cells indicated that this was indeed the case (Fig. 2)Citation . Densitometric analysis indicated that serum-starved Sparc -/- fibroblasts expressed {approx}12% of the level of IGFI-R found in wild-type fibroblasts. By contrast, we observed no difference in insulin receptor levels in both cell types (Fig. 2)Citation . To determine whether PDGF up-regulated expression of the IGFI-R in Sparc -/- cells, we examined the steady-state levels of IGFI-R message by Northern analysis in serum-starved cells treated with insulin as compared with insulin and PDGF. Consistent with the results of the Western blot analysis, steady-state levels of IGFI-R mRNA in SPARC -/- fibroblasts were significantly lower than in wild-type cells (Fig. 3A)Citation . Insulin treatment resulted in down-regulation of IGFI-R message in wild-type fibroblasts and had no discernible effect on the already low levels in SPARC -/- cells (Fig. 3A)Citation . In contrast, the addition of PDGF and insulin prevented down-regulation of IGFI-R transcripts in wild-type cells and dramatically up-regulated the level of IGFI-R message in SPARC -/- cells (Fig. 3B)Citation . We concluded that IGFI-R expression in wild-type fibroblasts was coordinately regulated by both endogenous SPARC and exogenous PDGF. SPARC expression was essential to IGFI-R expression when fibroblasts were deprived of exogenous growth factors. However, exogenous PDGF could compensate for the absence of SPARC by up-regulating IGFI-R expression in SPARC -/- cells.



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  		Fig. 2. Immunoblot analysis of the IGFI-R {alpha}-subunit and the insulin receptor ß-subunit proteins in serum-deprived SPARC-deficient (-/-) and wild-type (+/+) cells. A, results of Western blot analyses using antibodies to the respective receptors. MW, molecular weight. B, results of the densitometric analysis of the hybridization signals in A. Arbitrary units for signal intensity relative to background are shown.

 


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  		Fig. 3. Time-dependent changes in expression of steady-state IGFI-R transcripts in serum-deprived SPARC-deficient (-/-) and control (+/+) fibroblasts upon restimulation with either insulin alone (A) or with insulin and PDGF (B).

 
IGFI-dependent Expression of Cyclin A and tk Is Disrupted in Cells Lacking SPARC.
To assess the consequences of SPARC deficiency on IGFI-dependent cell cycle progression, we examined the expression of genes that are up-regulated during the G1 and S phases of the cell cycle. Important events during early G1 include activation of c-myc and cyclin D1 gene expression, followed by phosphorylation of pRb and its related family members (p107 and p130), collectively termed pocket proteins. Phosphorylation of the pocket proteins leads to the release and activation of the E2F family of transcription factors, which proceed to induce genes associated with the S-phase progression, for example, cyclin A and tk (26) . Consistent with impaired S-phase progression in insulin-treated Sparc -/- cells, expression of cyclin A and tk transcripts was markedly reduced compared with wild-type cells (Fig. 4A)Citation . Expression of cyclin A was abbreviated and undetectable at 18 and 24 h after insulin treatment, whereas expression of tk remained significantly reduced at all times. By comparison, activation of c-myc or cyclin D1 mRNA (data not shown) was only marginally affected. Because PDGF together with insulin restored both IGFI-R expression and DNA synthesis in SPARC -/- cells, we determined whether the combination of these two growth factors would also restore expression of cyclin A and tk transcripts. We observed that both messages were poorly expressed at 10 h in insulin-treated SPARC -/- and PDGF-treated wild-type cells (Fig. 4, B and C)Citation . PDGF alone caused late induction (32 h) of both cyclin A and tk transcripts in wild-type cells (Fig. 4C)Citation . However, cyclin A and tk mRNA levels were induced by PDGF and insulin treatment in Sparc -/- and wild-type cells at 20 h (Fig. 4B)Citation . Thus, PDGF treatment of SPARC -/- not only restored DNA synthesis and IGFI-R expression to wild-type levels but also up-regulated expression of the cyclin A and tk genes in response to insulin. Taken together, these results indicated that reduced cell cycle progression in SPARC -/- cells in response to insulin was attributable to reduced expression of the IGF-1R and reduced expression of cyclin A and tk.



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  		Fig. 4. Expression patterns of cell cycle regulatory genes in serum-deprived SPARC-deficient (-/-) and wild-type (+/+) cells at various time points after restimulation with either insulin alone (A) or with insulin and PDGF (B) and with PDGF alone in wild-type (+/+) cells (C).

 
SPARC Is Necessary for IGFI-R-dependent Induction of p107.
Transcriptional activation of the tk and cyclin A genes in S phase depends on E2F activation, which in turn depends upon phosphorylation and release of E2F from Rb family pocket proteins (27, 28, 29) . Therefore, to further define the mechanism by which proper induction of cyclin A and tk genes during S phase was disrupted in SPARC -/- cells, we compared the expression and phosphorylation of pocket proteins by Western blotting after treatment of cells with insulin. No difference was detected in either the expression or phosphorylation of Rb or p130 between SPARC -/- and wild-type cells. In contrast, we noted a marked reduction in the steady-state levels of p107 in SPARC -/- cells prior to and after insulin stimulation (Fig. 5A)Citation . This finding suggested that the defective activation of cyclin A and tk gene expression in SPARC -/- cells in response to IGFI-R stimulation might be related to loss of function in a specific p107/E2F complex. We concluded that SPARC regulated p107 levels through an IGFI-R-dependent pathway and that this event was associated with proper regulation of cyclin A and other S-phase progression genes such as tk. As in the case of the IGFI-R, cyclin A, and tk mRNA expression, the addition of PDGF to SPARC -/- cells restored p107 expression (Fig. 5B)Citation , underscoring the critical importance of sufficient IGFI-R activation in the normal expression of p107, cyclin A, and tk in this cell type.



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  		Fig. 5. Time-dependent expression and phosphorylation (arrow) patterns of pRb, p107, and p130 in serum-deprived SPARC-deficient (-/-) and control (+/+) cells after restimulation with insulin (A) and of p107 in serum-deprived SPARC-deficient (-/-) cells after restimulation with insulin alone or with insulin and PDGF (B).

 
Discussion

In this study, we defined a mechanism through which a component of the ECM influences cell cycle progression. The growth factor requirements of fibroblasts in culture have been extensively analyzed and so provide a fine model to probe the integrated regulation of adhesion and growth factor signaling, which remain an important area of investigation. Two categories of growth factors cooperate to stimulate fibroblast proliferation; these include the so-called competence and progression factors, typified by PDGF and IGFI (30, 31, 32, 33) . In its role as a progression factor, IGFI amplifies DNA synthesis in cells that have already been stimulated to enter G1 by treatment with competence factors such as PDGF. Whereas PDGF primarily regulates the G0-G1 entry, IGFI accelerates progression through G1 phase and into S phase. Although effects of IGFI on gene expression have been described (31) , it is poorly understood how IGFI influences G1 progression.

SPARC is a secreted protein deposited into fibroblast ECM and has been implicated in tissue remodeling and wound healing, as suggested by increased expression of SPARC at the edges of healing wounds (11) and in renal interstitial fibrosis (34) . SPARC associates with collagen (35) and/or other undefined cell surface molecules (19) , supporting the idea that SPARC contributes to signals induced by ECM molecules and is relevant to tissue remodeling. To better define physiological functions of SPARC, we generated mice that harbor a homozygous null mutation of the Sparc gene (9) . Although viable and developmentally normal, these mice exhibit significantly slower wound healing,4 consistent with a positive role for SPARC in cell growth. In the present study, we describe that embryonal fibroblasts from nullizygous mice have a cell cycle progression deficit related to impaired IGFI-R expression and attendant cell cycle regulatory defects.

Specifically, SPARC-deficient embryonal fibroblasts responded normally to stimulation of DNA synthesis by PDGF but poorly to stimulation by insulin. This defect was traced to a reduction in expression of the IGFI-R itself. When added together with insulin, PDGF restored expression of the IGFI-R as well as its attendant biological effects, indicating that PDGF can, at least partially, substitute for SPARC in these fibroblasts. To relate down-regulation of the IGFI-R in Sparc-nullizygous cells with specific cell cycle events, we examined the kinetics and levels of induction of four genes, the up-regulation of which accompanies G1-S transit. Up-regulation of c-myc and cyclin D1 transcripts occurs in early to mid G1, whereas cyclin A (36) and tk (37 , 38) are up-regulated during G1 exit and entry into S phase. Expression of c-myc and cyclin D1 mRNAs in response to IGFI-R stimulation was only marginally decreased in SPARC -/- cells when compared with controls. By contrast, cyclin A and tk messages were expressed at markedly low levels in SPARC-deficient cells, consistent with earlier studies demonstrating that IGFI acts in mid-to-late G1 to promote the G1-S transition (30, 31, 32) . At present, it is unknown how SPARC affects IGFI-R expression. SPARC may up-regulate IGFI-R expression through an integrin-dependent pathway because it binds collagen and is thought to mediate its effects in part by affecting collagen configuration and integrin-mediated focal adhesion (19 , 39) . This issue will be important to investigate in future work.

Increased expression of cyclin A and tk in late G1 and S has been shown to depend on cell cycle progression past the G1 restriction point and on the Rb family pocket proteins. Hyperphosphorylation of the pocket proteins leads to release of members of the E2F family of transcription factors, which contribute to transcription of the cyclin A and tk genes (27 , 40 , 41) . For this reason, we compared the levels and phosphorylation states of the three known pocket proteins in SPARC -/- and control wild-type fibroblasts. This analysis revealed a specific loss of p107 expression in SPARC -/- cells, whereas expression and phosphorylation of Rb and p130 were comparable with those observed in wild-type fibroblasts. Taken together, the results suggested that reduced signaling by the IGFI-R selectively affected expression of p107 but not Rb or p130.

Several recent studies have implicated p107 specifically in regulating cyclin A expression levels. For example, the cyclin A promoter contains a variant E2F site that binds E2F complexes containing p107 but not Rb (40) , and adenovirus E1A activates cyclin A gene transcription in the absence of growth factors via interactions with p107 but not Rb (42) . In addition, p107 affects cyclin A gene transcription primarily by interacting with E2F-4 and, possibly, E2F-5, but not with E2F-1, E2F-2, or E2F-3, which are bound preferentially to Rb (40 , 41 , 43) . Lastly, loss of free, transcriptionally active E2F upon genotoxic stress is attributable, at least in part, to the sequestration of E2F-4 by p107 (41) . If indeed unphosphorylated p107 can sequester E2F-4 and thus constrain transcriptional activation of the cyclin A gene (41 , 43) , the reduced expression of p107 in insulin-treated SPARC -/- fibroblasts should have resulted in more free E2F-4 and increased expression of cyclin A. However, we observed the opposite result. One possible explanation for this apparent paradox is that p107 is needed to translocate E2F-4 from the cytoplasm to the nucleus during late G1 and S phase (44, 45, 46) . Thus, reduced or absent expression of p107 may constrain transport of E2F-4 to the nucleus and prevent transcription of cyclin A. Alternatively, IGFI-R activation may regulate p107 and cyclin A expression by independent pathways. This alternative view is supported by the observation that cyclin A expression in serum-stimulated cells is unaffected by p107 loss (47) .

The postulated role of SPARC in cell adhesion invites a comparison between the cell cycle events observed in IGFI-R-stimulated Sparc -/- fibroblasts and those observed in fibroblasts denied ECM adhesion (29 , 48) . Interestingly, markedly similar reductions in cyclin A but not cyclin D1 levels occur in both experimental conditions. The present study links reduced cyclin A expression in SPARC -/- cells to reduced IGFI-R signaling capacity. Thus, one way by which ECM molecules ensure the anchorage dependence of fibroblast proliferation may be to control the expression of growth factor receptors. It will be important to investigate whether adhesion molecules other than SPARC serve to modulate growth factor receptor expression and growth factor responsiveness through similar mechanisms.

Materials and Methods

Isolation and Culture of Embryonic Fibroblasts.
A mixed genetic background (F2) of C57BL/6j x 129SvEv wild-type and SPARC-null mice (9) were maintained in a pathogen-free facility. Primary embryonic fibroblasts were prepared from embryos at 13.5 days of gestation. Briefly, embryos were mechanically disaggregated, and tissue fragments were incubated for 15 min in a solution of Versene containing 0.5% trypsin at 37°C. Trypsin was inactivated with two volumes of DMEM [Life Technologies Inc. (LTI), Gaithersburg, MD] containing 10% FBS (Hyclone, Logan, UT). Large tissue fragments were separated from small cell aggregates by gravitational sedimentation for 20–30 min at room temperature. The supernatant was carefully removed, and small cell aggregates were collected by centrifugation. After several washes with DMEM containing 10% FBS, penicillin (10 IU/ml; LTI, Grand Island, NY), streptomycin (10 µg/ml; LTI), and fungizone (0.25 µg/ml; LTI), cells were reseeded on tissue culture-treated plastic and propagated in DMEM containing 10% FBS (referred to as growth medium). In all experiments, wild-type and SPARC-null embryonic fibroblasts at identical passage numbers (<8) were used.

Purification of Mouse SPARC.
Mouse SPARC was purified as described previously with minor modifications (6 , 35) . Briefly, a 50% ammonium sulfate precipitate from mouse PYS-2 cell culture supernatant was dissolved and dialyzed against 4 M urea, 50 mM Tris-HCl (pH 8.0). The fraction containing SPARC and albumin was retrieved on a DEAE-cellulose (Amersham Pharmacia, Piscataway, NJ) column by step gradient elution at 175 mM NaCl. Albumin was removed by dialysis against distilled water for 48 h, which led to SPARC precipitation. The precipitate was dissolved in 2 M urea, 20 mM Tris-HCl (pH 8.0) and passed through a Mono Q column (Amersham Pharmacia) equilibrated in the same buffer. SPARC was eluted using a linear 0–0.6 M NaCl gradient on an FPLC column (Amersham Pharmacia). The fractions eluted between 0.3 and 0.36 M salt were passed three more times through the column, dialyzed against 0.2 M ammonium bicarbonate, and lyophilized. The lyophilized material was dissolved in phosphate-buffer saline containing 2 mM CaCl2 and stored at -80°C. Purity of the SPARC preparation was verified by Western blot analysis using anti-SPARC antibody (49) .

Incorporation of [3H]Thymidine.
Cells were seeded in 96-well plates (1 x 104 cells/well) and incubated overnight in growth medium followed by a 24-h incubation in serum-free DMEM. Stimulation of quiescent cells with growth factors was achieved in serum-free DMEM supplemented with human serum albumin (5 mg/ml; Sigma Chemical Co., St. Louis, MO) and transferrin (2.5 µg/ml; Sigma). DNA synthesis was determined after addition of insulin (2 µg/ml; Sigma), PDGF-AB (10 ng/ml; LTI), SPARC protein (20 µg/ml), or 10% FBS. In continuous labeling experiments, [3H]thymidine (1 µCi/ml; NEN, Boston, MA) was added together with the growth factors for time periods varying between 0 and 40 h. To determine the time-dependent S-phase entry, serum-starved cells were pretreated for various times with growth factors, followed by pulse labeling with [3H]thymidine (10 µCi/ml) for 1 h immediately before harvest. DNA-bound [3H]thymidine was immobilized on filter paper with the aid of an automated cell harvester (Tomtec, Orange, CT), and the tritium incorporation was determined by scintillation counting.

Propidium Iodide Staining of DNA.
Serum-starved, quiescent cells were treated with growth factors for 20 h as described, fixed in 70% ethanol, and stained with 10 µg/ml propidium iodide in PBS. Propidium iodide uptake was analyzed using a Coulter/EPICS XL-MCL FACScan.

Northern Blot Analysis.
Primary fibroblasts were seeded at {approx}50–60% confluency in 150-mm2 tissue culture plates in growth medium for 16 h. After removal of medium, attached cells were washed twice and further incubated for 72 h in serum-free DMEM containing albumin as the sole exogenous protein. Serum-starved cells were then freshly exposed to DMEM containing albumin and transferrin and supplemented with either 2 µg/ml insulin or 10 ng/ml PDGF or 10% FBS. Duplicate plates were harvested and processed for RNA extraction at several time points as indicated. Total RNA was isolated by guanidine thiocyanate extraction and analyzed by Northern blotting. Hybridization probes were prepared using a random primed polymerization kit with [32P]dCTP (NEN). cDNA fragments used to prepare the probes were derived from c-myc (50) , cyclin A (51) , tk (52) , L32 (53) , and the IGFI-R (54) .

Immunoprecipitation and Western Blot Analysis.
Total protein was extracted using 1% NP40, 150 mM KCl, and 20 mM HEPES (pH 7.8) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 2.5 mg/ml aprotinin, 0.25 mg/ml pepstatin, and 2.5 units/ml leupeptin). Cell extracts, with equal amounts of protein from all samples, were fractionated on 7% SDS-PAGE gels and transferred to nitrocellulose (MSI, Westborough, MA). Blots were probed with anti-pRb antibody C-15 (Santa Cruz Biotechnology, Santa Cruz, CA) or with rabbit antibodies raised to p130 and p107 (gifts from Dr. A. Giordano, Thomas Jefferson University). The blots were developed using appropriate horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence kit (Pierce, Rockford, IL).

Receptor proteins for insulin and IGFI were partially purified from fibroblast extracts prepared in 1% Triton X-100, 150 mM NaCl, and 50 mM HEPES (pH 7.6) using a wheat germ agglutinin-agarose column (Sigma). Bound proteins were eluted from the column with 0.45 M n-acetylglucosamine (Sigma) in the same buffer, and the eluates were concentrated using Centricon-10 filters (Amicon, Beverly, MA). Western blots were made from the equal amount of partially purified proteins from Sparc -/- and wild-type cells, probed with an anti-IGFI-R {alpha}-subunit antibody (Upstate Biotechnology, Lake Placid, NY), and developed as above. The same blots were stripped by three washes in Tris-buffered saline containing 0.5% Tween 20 and then probed with the anti-insulin receptor ß-subunit antibody (Transduction Lab, Lexington, KY). The blots were developed as before, except that an alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) method was used. The experiment was repeated twice.

Acknowledgments

We thank A. Giordano (Thomas Jefferson University, Philadelphia, PA) for providing antibodies to p130/Rb2 and p107. A. B. acknowledges the support and encouragement from Dr. Giovanni Rovera, the Director of The Wistar Institute.

Footnotes

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

1 Supported by National Science Foundation Grant EEC9814404 (to C. C. H.), NIH Grants CA81008 (to U. R.) and CA65892 (to G. C. P.), and by The Wistar Institute funds. Back

2 To whom requests for reprints should be addressed, at The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: (215) 898-3796; Fax: (215) 898-3868; E-mail: Chowe{at}wistar.upenn.edu Back

3 The abbreviations used are: SPARC, secreted protein acidic and rich in cysteine; ECM, extracellular matrix; IGFI, insulin-like growth factor I; IGFI-R, IGFI receptor; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; tk, thymidine kinase; pRb, retinoblastoma protein. Back

4 A. Basu and C. C. Howe, unpublished results. Back

Received for publication 11/20/98. Revision received 8/26/99. Accepted for publication 9/ 1/99.

References

  1. Mason I. J., Murphy D., Munke M., Francke U., Elliott R. W., Hogan B. L. Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell "culture shock" glycoprotein of Mr 43,000. EMBO J., 5: 1465-1472, 1986.[Medline]
  2. Lankat-Buttgereit B., Mann K., Deutzmann R., Timpl R., Krieg T. Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin). FEBS Lett., 236: 352-356, 1988.[Medline]
  3. McVey J. H., Nomura S., Kelly P., Mason I. J., Hogan B. L. Characterization of the mouse SPARC/osteonectin gene. Intron/exon organization and an unusual promoter region. J. Biol. Chem., 263: 11111-11116, 1988.[Abstract/Free Full Text]
  4. Bornstein P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol., 130: 503-506, 1995.[Free Full Text]
  5. Lane T. F., Sage E. H. The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J., 8: 163-173, 1994.[Abstract]
  6. Iruela-Arispe M. L., Vernon R. B., Wu H., Jaenisch R., Sage E. H. Type I collagen-deficient Mov-13 mice do not retain SPARC in the extracellular matrix: implications for fibroblast function. Dev. Dyn., 207: 171-183, 1996.[Medline]
  7. Sage H., Vernon R. B., Decker J., Funk S., Iruela-Arispe M. L. Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J. Histochem. Cytochem., 37: 819-829, 1989.[Abstract]
  8. Gilmour D. T., Lyon G. J., Carlton M. B. L., Sanes J. R., Cunningham J. M., Anderson J. R., Hogan B. L. M., Evans M. J., Colledge W. H. Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J., 17: 1860-1870, 1998.[Medline]
  9. Norose K., Clark J. I., Syed N. A., Basu A., Heber-Katz E., Sage E. H., Howe C. C. SPARC deficiency leads to early onset cataractogenesis. Invest. Ophthalmol. Vis. Sci., 39: 2674-2680, 1998.[Abstract/Free Full Text]
  10. Porter P. L., Sage E. H., Lane T. F., Funk S. E., Gown A. M. Distribution of SPARC in normal and neoplastic human tissue. J. Histochem. Cytochem., 43: 791-800, 1995.[Abstract]
  11. Reed M. J., Puolakkainen P., Lane T. F., Dickerson D., Bornstein P., Sage E. H. Differential expression of SPARC and thrombospondin 1 in wound repair: immunolocalization and in situ hybridization. J. Histochem. Cytochem., 41: 1467-1477, 1993.[Abstract]
  12. Ledda F., Bravo A. I., Adris S., Bover L., Mordoh J., Podhajcer O. L. The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma. J. Investig. Dermatol., 108: 210-214, 1997.[Medline]
  13. Ledda M. F., Adris S., Bravo A. I., Kairiyama C., Bover L., Chernajovsky Y., Mordoh J., Podhajcer O. L. Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells. Nat. Med., 3: 171-176, 1997.[Medline]
  14. Mok S. C., Chan W. Y., Wong K. K., Muto M. G., Berkowitz R. S. SPARC, an extracellular matrix protein with tumor-suppressing activity in human ovarian epithelial cells. Oncogene, 12: 1895-1901, 1996.[Medline]
  15. Funk S. E., Sage E. H. The Ca2+-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells. Proc. Natl. Acad. Sci. USA, 88: 2648-2652, 1991.[Abstract/Free Full Text]
  16. Funk S. E., Sage E. H. Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts. J. Cell. Physiol., 154: 53-63, 1993.[Medline]
  17. Sage H., Decker J., Funk S., Chow M. SPARC: a Ca2+-binding extracellular protein associated with endothelial cell injury and proliferation. J. Mol. Cell. Cardiol., 1: 13-22, 1989.
  18. Goldblum S. E., Ding X., Funk S. E., Sage E. H. SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function. Proc. Natl. Acad. Sci. USA, 91: 3448-3452, 1994.[Abstract/Free Full Text]
  19. Murphy-Ullrich J. E., Lane T. F., Pallero M. A., Sage E. H. SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca2+-binding EF-hand. J. Cell. Biochem., 57: 341-350, 1995.[Medline]
  20. Scher C. D., Shepard R. C., Antoniades H. N., Stiles C. D. Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim. Biophys. Acta, 560: 217-241, 1979.[Medline]
  21. Stiles C. D., Pledger W. J., Tucker R. W., Martin R. G., Scher C. D. Regulation of the Balb/c-3T3 cell cycle-effects of growth factors. J. Supramol. Struct., 13: 489-499, 1980.[Medline]
  22. Furlanetto R. W., DiCarlo J. N. Somatomedin-C receptors and growth effects in human breast cells maintained in long-term tissue culture. Cancer Res., 44: 2122-2128, 1984.[Abstract/Free Full Text]
  23. Kamihagi K., Katayama M., Ouchi R., Kato I. Osteonectin/SPARC regulates cellular secretion rates of fibronectin and laminin extracellular matrix proteins. Biochem. Biophys. Res. Commun., 200: 423-428, 1994.[Medline]
  24. Rubini M., Werner H., Gandini E., Roberts C. T., Jr., LeRoith D., Baserga R. Platelet-derived growth factor increases the activity of the promoter of the insulin-like growth factor-1 (IGF-1) receptor gene. Exp. Cell Res., 211: 374-379, 1994.[Medline]
  25. Ambrose D., Resnicoff M., Coppola D., Sell C., Miura M., Jameson S., Baserga R., Rubin R. Growth regulation of human glioblastoma T98G cells by insulin-like growth factor-1 and its receptor. J. Cell. Physiol., 159: 92-100, 1994.[Medline]
  26. Coppock D. L., Pardee A. B. Control of thymidine kinase mRNA during the cell cycle. Mol. Cell. Biol., 7: 2925-2932, 1987.[Abstract/Free Full Text]
  27. Dou Q. P., Zhao S., Levin A. H., Wang J., Helin K., Pardee A. B. G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter. J. Biol. Chem., 269: 1306-1313, 1994.[Abstract/Free Full Text]
  28. Good L., Chen J., Chen K. Y. Analysis of sequence-specific binding activity of cis-elements in human thymidine kinase gene promoter during G1/S phase transition. J. Cell. Physiol., 163: 636-644, 1995.[Medline]
  29. Shulze A., Zerfass-Thome K., Berges J., Middendorp S., Jansen-Durr P., Henglein B. Anchorage-dependent transcription of the cyclin A gene. Mol. Cell. Biol., 16: 4632-4638, 1996.[Abstract]
  30. Bitterman P. B., Rennard S. I., Adelberg S., Crystal R. G. Role of fibronectin as a growth factor for fibroblasts. J. Cell Biol., 97: 1925-1932, 1983.[Abstract/Free Full Text]
  31. Campisi J., Pardee A. B. Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor. Mol. Cell. Biol., 4: 1807-1814, 1984.[Abstract/Free Full Text]
  32. Pardee A. B., Coppock D. L., Yang H. C. Regulation of cell proliferation at the onset of DNA synthesis. J. Cell Sci. Suppl., 4: 171-180, 1986.[Medline]
  33. Chen Y., Rabinovitch P. S. Platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor I regulate specific cell-cycle parameters of human diploid fibroblasts in serum-free culture. J. Cell. Physiol., 140: 59-67, 1989.[Medline]
  34. Pichler R. H., Hugo C., Shankland S. J., Reed M. J., Bassuk J. A., Andoh T. F., Lombardi D. M., Schwartz S. M., Bennett W. M., Alpers C. E., Sage E. H., Johnson R. J., Couser W. G. SPARC is expressed in renal interstitial fibrosis and in renal vascular injury. Kidney Int., 50: 1978-1989, 1996.[Medline]
  35. Mayer U., Aumailley M., Mann K., Timpl R., Engel J. Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV. Eur. J. Biochem., 198: 141-150, 1991.[Medline]
  36. Pines J., Hunter T. Cyclins A and B1 in the human cell cycle. Ciba Found. Symp., 170: 187-196, 1992.[Medline]
  37. Schlosser C. A., Steglich C., deWet J. R., Scheffler I. E. Cell cycle-dependent regulation of thymidine kinase activity introduced into mouse LMTK- cells by DNA and chromatin-mediated gene transfer. Proc. Natl. Acad. Sci. USA, 78: 1119-1123, 1981.[Abstract/Free Full Text]
  38. Bradshaw H., Jr. Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene. Proc. Natl. Acad. Sci. USA, 80: 5588-5591, 1983.[Abstract/Free Full Text]
  39. Motamed K., Sage E. H. SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation dependent pathway. J. Cell. Biochem., 70: 543-552, 1998.[Medline]
  40. Schulze A., Zerfass K., Spitkovsky D., Middendorp S., Berges J., Helin K., Jansen-Durr P., Henglein B. Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site. Proc. Natl. Acad. Sci. USA, 92: 11264-11268, 1995.[Abstract/Free Full Text]
  41. Spitkovsky D., Schulze A., Boye B., Jansen-Durr P. Down-regulation of cyclin A gene expression upon genotoxic stress correlates with reduced binding of free E2F to the promoter. Cell Growth Differ., 8: 699-710, 1997.[Abstract]
  42. Zerfass K., Spitkovsky D., Schulze A., Joswig S., Henglein B., Jansen-Durr P. Adenovirus E1A activates cyclin A gene transcription in the absence of growth factors through interaction with p107. J. Virol., 70: 2637-2642, 1996.[Abstract]
  43. Xiao Z. X., Ginsberg D., Ewen M., Livingston D. M. Regulation of the retinoblastoma protein-related protein p107 by G1 cyclin-associated kinases. Proc. Natl. Acad. Sci. USA, 93: 4633-4637, 1996.[Abstract/Free Full Text]
  44. Lindeman G. J., Gaubatz S., Livingston D. M., Ginsberg D. The subcellular localization of E2F-4 is cell-cycle dependent. Proc. Natl. Acad. Sci. USA, 94: 5095-5100, 1997.[Abstract/Free Full Text]
  45. Verona R., Moberg K., Estes S., Starz M., Vernon J. P., Lees J. A. E2F activity is regulated by cell cycle-dependent changes in subcellular localization. Mol. Cell. Biol., 17: 7268-7282, 1997.[Abstract]
  46. Magae J., Wu C. L., Illenye S., Harlow E., Heintz N. H. Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members. J. Cell Sci., 109: 1717-1726, 1996.[Abstract]
  47. Hurford R. K., Jr., Cobrinik D., Lee M. H., Dyson N. pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes. Genes Dev., 11: 1447-1463, 1997.[Abstract/Free Full Text]
  48. Guadagno T. M., Ohtsubo M., Roberts J. M., Assoian R. K. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science (Washington DC), 262: 1572-1575, 1993.[Abstract/Free Full Text]
  49. Latham K. E., Howe C. C. SPARC synthesis in pre-implantation and early post-implantation embryos. Roux’s Arch. Dev. Biol., 199: 364-369, 1991.
  50. Prendergast G. C., Lawe D., Ziff E. B. Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation. Cell, 65: 395-407, 1991.[Medline]
  51. Wang J., Chenivesse X., Henglein B., Brechot C. Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature (Lond.), 343: 555-557, 1990.[Medline]
  52. Lin P. F., Lieberman H. B., Yeh D. B., Xu T., Zhao S. Y., Ruddle F. H. Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences. Mol. Cell. Biol., 5: 3149-3156, 1985.[Abstract/Free Full Text]
  53. Rajchel A., Chan Y. L., Wool I. G. The primary structure of rat ribosomal protein L32. Nucleic Acids Res., 16: 2347 1988.[Free Full Text]
  54. Wada J., Liu Z. Z., Alvares K., Kumar A., Wallner E., Makino H., Kanwar Y. S. Cloning of cDNA for the {alpha} subunit of mouse insulin-like growth factor I receptor and the role of the receptor in metanephric development. Proc. Natl. Acad. Sci. USA, 90: 10360-10364, 1993.[Abstract/Free Full Text]



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