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Keywords:

  • growth hormone;
  • osteoblast;
  • signal transduction;
  • transcription factor;
  • DNA binding

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) are important growth factors for postnatal longitudinal bone growth. Although many effects of GH on bone growth are mediated by IGF-1, GH can directly influence bone cells. Limited knowledge exists regarding specific intracellular signaling pathways and genes activated by GH in bone cells. GH is known to activate several intracellular signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and both isoforms of STAT5, A and B. STAT5 gene deletion experiments have shown the importance of these transcription factors for growth. To understand the molecular mechanism(s) behind this, different experimental models are needed. The UMR 106 cell line is a rat clonal osteosarcoma cell line with osteoblast-like phenotypic properties, one is the endogenous expression of GH receptor (GHR). The present study focused on whether these cells express a functional GH-responsive JAK2/STAT5 pathway. Analysis of cell extracts by immunoprecipitation and Western blot showed that physiological concentrations of GH activated JAK2. Western blot analysis of nuclear extracts from GH-stimulated UMR 106 cells showed that physiological concentrations of GH induced nuclear translocation of both STAT5 isoforms, but with STAT5A being predominant. Both isoforms displayed similar nuclear turnover after GH stimulation of cells. Gel electrophoretic mobility shift assay (GEMSA) of nuclear extract revealed that both STAT5A and STAT5B obtained DNA-binding capacity after GH stimulation. Thus, we have shown, for the first time, the expression and GH-induced activation of JAK2 and STAT5A/B in UMR 106 osteoblast-like cells. This study also shows that this cell line is a suitable experimental model to study unique GH effects in osteoblasts mediated by STAT5.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

GROWTH HORMONE (GH) and insulin-like growth factor 1 (IGF-1) are the most important growth factors for normal postnatal longitudinal bone growth.(1,2) IGF-1 is produced mainly by the liver under the influence of GH but also is synthesized locally by many tissues in the body. In bone cells, GH induces expression of IGF-1. Although many effects of GH on bone growth are mediated by IGF-1, experimental data indicate that GH can directly influence bone cell function.(2) The intracellular mechanism involved in these GH-specific effects on bone cells is at present not fully understood. Furthermore, there is a lack of knowledge regarding osteoblast genes regulated by GH.

Earlier studies have shown that GH activates different intracellular signaling pathways.(3) GH binding to a membrane-bound GH receptor (GHR) mediates these cellular effects.(3) GHR binding activates receptor-bound members of the Janus family of tyrosine kinases (JAK). In most cases GH activates JAK2. Activated JAK2 tyrosine phosphorylates and activates several intracellular signal mediators, including members of the signal transducers and activators of transcription (STAT) family of transcription factors. After activation, STATs translocate to the nucleus, bind DNA, and regulate gene transcription. At present, seven mammalian STAT genes have been identified.(4,5)

GH mainly activates STAT5 but in experimental models it also has been shown to activate STAT1 and STAT3.(6,7) STAT5 was first identified as a mammary gland factor required for prolactin (PRL)-stimulated transcription of milk protein genes.(8) STAT5 was later found to be present in several other tissues and to be activated by other hormones including GH, growth factors, and cytokines.(9,10)

Two forms of STAT5 (STAT5A and STAT5B), encoded by two different genes, have been identified and are shown to be expressed in most tissues.(11,12) The functional significance of the presence of the two forms is at present not fully understood. Although overlapping functions seem to exist, recent animal studies have indicated unique functions for each STAT isoform. Genetic disruption of the STAT5A gene in mice resulted in impaired terminal differentiation of the mammary glands during pregnancy, resulting in lack of milk production.(13) A key regulator of these two processes is PRL. Deletion of STAT5B gene resulted in reduced body growth of male mice, a process of which GH is a key regulator.(14,15) The simultaneous deletion of both genes resulted in the most severe growth and reproductive defects, revealing functional redundancy of the STAT5 proteins in physiological processes mediated by GHR and PRL.(15)

As described previously, the direct cellular effects of GH on bone cells are not fully understood. In addition, the GH-activated intracellular signaling pathways and GH-activated genes in these cells are not fully described. Relevant experimental models are needed to gain insight into these issues. GH has been shown to stimulate proliferation of fetal chicken and mouse osteoblasts, rat osteoblast-like cells, and osteoblast precursor cells derived from human bone marrow stroma.(16–19) Functional GHR has been described in rat and human bone osteoblast-like cell lines.(18,20) The recent findings using gene deletion experiments motivated us to search for an in vitro experimental model suitable for investigating GH activation of STAT5 pathway.

In this study we have investigated the presence of the JAK2/STAT5 signaling system as well as its GH-activated characteristics in the rat osteoblast-like osteosarcoma cell line UMR 106. This cell line shares a number of phenotypic properties with mature osteoblasts; one of them is the endogenous expression of the GHR.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Cell culture

Rat osteosarcoma cells UMR 106 were routinely grown in monolayer culture at 37°C in 5% CO2-air in modified essential medium (MEM; Life Technologies, Ltd., Paisley, Scotland) supplemented with 5% fetal bovine serum (FBS; Life Technologies), 1 mM L-glutamine, and 50 U/ml penicillin (Life Technologies).

Preparation of nuclear extract

The cells were cultured as described previously. Before addition of bovine GH (bGH), cells were starved of fetal calf serum for 12–16 h. After treatment with bGH, cells were cooled on ice and rinsed with ice-cold phosphate-buffered saline (PBS). Cells were then scraped into 10 ml of extraction buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 6 mM MgCl2, 1 mM dithiothreitol [DTT], and 0.1 mM Na3VO4) and disrupted with a Dounce homogenizer (Götenborgstermometerfabrik, Gothenburg, Sweden). The nuclear pellet obtained after centrifugation was resuspended in 3 vol of lysis buffer (20% glycerol, 20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM DTT, and 0.1 mM Na3VO4) and incubated on ice for 30 minutes. The supernatant obtained after centrifugation was used as nuclear extract. Protein concentration was measured by the Bradford method. The nuclear extract obtained was further analyzed with Western blotting or gel electrophoretic mobility shift assay (GEMSA).

Preparation of whole cell extract and immunoprecipitation

UMR 106 cells were grown in 10-cm plates and before addition of bGH, cells were starved of fetal calf serum for 12–16 h. After treatment with 10 nM bGH for 5, 10, and 15 minutes, cells were rinsed with ice-cold PBS and scraped into lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1mM EDTA, 1mM DTT, 10 μg/μl leupeptin, 10 μg/μl pepstatin, 0.2 μg/μl aprotinin, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, and 1% Triton X-100) and kept on ice for 20 minutes. Then, the cell lysates were clarified by centrifugation at 4°C for 10 minutes at 13,000 rpm in a table-top centrifuge. Extracts (1 mg total protein) were immunoprecipitated overnight at 4°C with 3 μl anti-JAK 2 antibody (diluted 1:2000; Upstate Biotechnology, Lake Placid, NY, U.S.A.) and 50 μl protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) in lysis buffer. The immunoprecipitate was then washed twice with 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl (Tris-buffered saline [TBS]). Successful JAK2 precipitation and the phosphorylation status of JAK2 were analyzed by Western blotting with anti-JAK2 and antiphosphotyrosine antibodies (diluted 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), respectively, as described in the following.

Western blotting

Sodiumdodecyl sulfate (SDS)-solubilizing buffer was added to nuclear extract (50 μg) and samples were boiled. Proteins were separated on a 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, U.S.A.) by semidry blotting. The membrane was blocked for 1 h with a buffer containing 1% milk protein or, in the case of Western Blotting with antiphosphotyrosine antibody, 1% albumin in TBS. After washing, the membrane was incubated for 1 h with rabbit anti-STAT5 (diluted 1:400; Oncogene Research product, Calbiochem, Cambridge, MA, U.S.A.), rabbit anti-STAT5A (diluted 1:750; Santa Cruz Biotechnology), rabbit anti- STAT5B (diluted 1:1000; Upstate Biotechnology, Inc.), rabbit anti-JAK2 (diluted 1:2000; Upstate Biotechnology), or mouse antiphosphotyrosine (diluted 1:1000; Santa Cruz Biotechnology) in 1% milk protein/TBS plus 0.05% Tween 20 (TTBS). The secondary antibody, goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG coupled with horseradish peroxidase, diluted 1:5000 in TTBS, was applied for 1 h after washing the membrane three times with TTBS. The membrane was then analyzed with enhanced chemiluminescence (ECL) method (Amersham).

GEMSA

GEMSA was performed according to standard protocols.(21) Nuclear extracts were incubated with32P-labeled double-stranded β-casein oligonucleotides (TGCTTCTTGGAATT) in 15 μl of a buffer containing 4% Ficoll, 12 mM HEPES, pH 7.9, 4 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 1mM DTT, and 5 μg poly (dI-dC; Amersham Pharmacia Biotech). For supershift analysis chicken anti-sheep STAT5,(22) rabbit-anti STAT5A (Santa Cruz Biotechnology) and rabbit anti-STAT5B (Upstate Biotechnology, Inc.) were added to the incubation mixture before separation on polyacrylamide gel.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

GH-induced activation of JAK2 and nuclear translocation of STAT5A and STAT5B in UMR 106 osteoblast-like osteosarcoma cells

The presence of a GHR has been described earlier in UMR 106 osteoblast-like osteosarcoma cells.(18) To analyze if these cells also express JAK/STAT-signaling pathway, whether GH activates this system, and to see if both STAT5A and STAT5B are present, whole cell and nuclear extracts were prepared from GH-treated UMR 106 osteosarcoma cells. Whole cell extracts were immunoprecipitated with anti-JAK2 antibody. Immunoprecipitates were analyzed by Western blot technique using antiphosphotyrosine antibody. In Fig. 1 it can be seen that stimulation of UMR 106 cells with 10 nM bGH for 5, 10, and 15 minutes resulted in activation of JAK2. Nuclear extracts were analyzed by Western blot technique using antibodies that recognize both isoforms of STAT5 (STAT5A and STAT5B, Fig. 2A) or the unique C-terminal ends of STAT5A and STAT5B (Figs. 2B–2D). UMR 106 cells were first incubated with different concentrations of GH for 10 minutes and nuclear extracts were analyzed by Western blot (Figs. 2A–2C, lanes 1–5). This revealed that a 10-minute pulse with as low a concentration as 1 nM GH induced a clear nuclear translocation of both STAT5A and STAT5B protein. In Fig. 2A, the upper and lower immunoreactive species represent STAT5A (apparent Mr 96,000) and STAT5B (apparent Mr 93,000), respectively. Maximal effect was reached at 10 nM GH with no increase in STAT5 protein levels in nuclear extracts from cells treated with 100 nM and 1000 nM GH (Figs. 2A–2C, lanes 3–5). Weak immunoreactivity also was seen in unstimulated cells. At all GH concentrations tested, a higher amount of STAT5A was detected as compared with STAT5B (Fig. 2A, lanes 2–5).

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Figure Figure 1. JAK2 was immunoprecipitated from lysates of control and bGH-treated UMR 106 osteosarcoma cells with anti-JAK2 antibody. The phosphorylation status of the precipitated JAK2 was analyzed by Western blot with antiphosphotyrosine antibody. Successful and even precipitation of JAK2 was verified by Western blotting of stripped membrane with anti-JAK2 antibody.

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Figure Figure 2. GH induced nuclear translocation of STAT5 in UMR 106 osteoblast-like cells. Cells were grown in serum-containing medium and before addition of GH, the cells were starved of fetal calf serum for 12–16 h. The cells were treated with (A-C, lanes 1–5) GH at different concentrations for 10 minutes or with (A and B, lanes 6–12, and D, lanes 1–7) 10 nM GH for different time periods. Nuclear extracts were prepared as described in the Materials and Methods section, and proteins were separated by SDS-PAGE and analyzed by Western blotting with antibodies that recognize both isoforms of (A) STAT5, (B) STAT5A, or (C and D) STAT5B.

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Next, we analyzed the time required for the first appearance of STAT5 protein in the nucleus and the duration of STAT5 protein presence in the nucleus after a single stimulation of UMR 106 with GH. In this experiment the cells were stimulated with 10 nM GH for different times (Figs. 2A and 2B, lanes 6–12, and Fig. 2D, lanes 1–7). After 5 minutes both STAT5A and STAT5B were clearly present in nuclear extracts. A slight increase in protein levels was seen at 10 minutes, with STAT5A being the most prominent immunoreactive species (Fig. 2A, lane 8). At 30 minutes, a marked anti-STAT5 protein immunoreactive species with a slightly lower electrophoretic mobility was detected (Fig. 2A, lane 9). This could have been caused by an increased amount of STAT5A protein. Analysis of nuclear extract, taken from the same time point, with anti-STAT5A and anti-STAT5B antibodies revealed a lower electrophoretic mobility for both isoforms (Fig. 2B, lane 9, and Fig. 2D, lane 4). Thus, the increase in signal seen in Fig. 2A also could have been caused by too low a resolution of STAT5 isoforms. STAT5A and STAT5B protein levels were markedly decreased at 60 minutes and had returned to levels comparable with unstimulated cells at 120 minutes. Also, in this experiment low but detectable signals were seen for STAT5A and STAT5B in nuclear extracts from unstimulated cells.

STAT5A and STAT5B DNA-binding activity in nuclear extracts from GH-treated UMR 106 cells

The DNA-binding activity of STAT5 in nuclear extracts from GH-treated UMR 106 cells was analyzed with GEMSA using a radioactive labeled probe containing the proximal STAT5-binding site in the β-casein promoter. Nuclear extracts also were incubated with STAT5 antibody in order to determine the presence of STAT5 in gel-shifted protein-DNA complex.

UMR 106 cells were treated first with different concentrations of GH for 10 minutes (Fig. 3A). No DNA-binding activity was seen in nuclear extracts from untreated cells (Fig. 3A, lanes 1 and 2). GH treatment resulted in the appearance of a gel-shifted complex, which was already seen at 1 nM GH treatment and reached its maximum at 10 nM (Fig. 3A, lanes 3–6). A slight decrease in level of this complex was seen in nuclear extracts from cells treated with 100 nM and 1000 nM GH. A clear supershift was seen when nuclear extracts were incubated with STAT5 antibody showing that the gel-shifted complex contained STAT5 (Fig. 3A, lanes 4, 6, 8, and 10).

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Figure Figure 3. Gel electrophoretic shift analysis of nuclear extracts of GH-treated UMR 106 osteoblast-like cells. UMR 106 cells were grown as described in the Material and Methods section and in Fig. 2. The cells were treated with (A) GH at different concentrations for 10 minutes or with (B) 10 nM GH for different time periods. Nuclear extracts were prepared as described in the Materials and Methods section and incubated with [32P]-labeled β-casein probe and with or without anti-STAT5 and thereafter analyzed by GEMSA.

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To study the time course of GH-activated nuclear STAT5 with DNA-binding activity, UMR 106 cells were treated with 10 nM GH for different times (Fig. 3B). Also, in this experiment, no STAT5 DNA-binding activity was seen in untreated cells (Fig. 3B, lanes 1 and 2). STAT5 with DNA-binding activity was evident after 5 minutes of GH treatment and increased at 10 minutes of stimulation after which it slowly decreased up to 120 minutes of stimulation.

Because both STAT5A and STAT5B were shown by Western blot to be present in UMR 106 nuclear extracts, we investigated whether both isoforms had DNA-binding activity (Fig. 4). Nuclear extracts from UMR 106 cells, untreated or treated with 10 nM GH for 10 minutes, were incubated with a radiolabeled probe and with or without anti-STAT5, anti-STAT5A, or anti-STAT5B antibodies and thereafter analyzed by GEMSA. No DNA-binding activity was seen in untreated cells (Fig. 4A, lanes 1–3, and Fig. 4B, lanes 1 and 2). GH treatment induced a band, which was supershifted by the three antibodies (Fig. 4A, lanes 5 and 6, and Fig. 4B, lanes 4 and 5). This showed that GH activated both STAT5A and STAT5B in UMR 106 cells to DNA-binding form.

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Figure Figure 4. Supershift analysis of DNA-protein complexes in nuclear extracts from GH-treated UMR 106 osteoblast-like cells. UMR 106 cells were grown as described in the Material and Methods section and in Fig. 2. (A and B) Cells were treated with 10 nM GH for 10 minutes. Nuclear extracts were prepared as described in the Materials and Methods section and incubated with [32P]-labeled β-casein probe and with or without anti-STAT5, anti-STAT5A, or anti-STAT5B antibodies and thereafter analyzed by GEMSA.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The recently reported findings from STAT5 gene deletion experiments showing the importance of this signaling pathway for growth motivated us to study the molecular details of GH signaling in bone cells. The UMR 106 is a rat clonal cell line with well-characterized osteoblast-like phenotypic properties and has been shown to be a good in vitro cellular system for studies of the physiology of osteoblasts. It shares a number of phenotypic properties with mature osteoblasts. These similarities include an osteoblast-like morphological appearance, responsiveness to calciotropic agents such as parathyroid hormone(23,24) and vitamin D3,(25) and a relatively high level of expression of the cell surface alkaline phosphatase activity.(23) Additionally, UMR cells synthesize several matrix proteins expressed by normal osteoblasts including type 1 collagen(24) and proteoglycans.(26) Other characterized phenotypic properties are the expression of GHR; production of IGF-1; and response to the proliferative effects of GH, IGF-1, and insulin.(18,27,28) In the present study, we have analyzed if these cells express JAK2 and STAT5 and also, if present, if these signal mediators are activated by GH. Our experiments showed that UMR 106 cells express JAK2 and both isoforms of STAT5 (STAT5A and STAT5B) and that these are activated by GH.

Maximal nuclear translocation of both STAT5A and STAT5B and maximal activation of STAT5 to DNA-binding form in UMR 106 cells were obtained with 10 nM of GH, which is equivalent to 220 ng/ml and which is within the physiological range of GH concentration in vivo.(29) Higher concentrations of GH did not further increase nor decrease nuclear translocation or activation of STAT5 to a DNA-binding form. Thus, a biphasic “bell-shaped” dose-response curve for GHR activation, which has been described in some earlier studies,(30) could not be seen with UMR 106 cells. This experiment also showed that STAT5A was the predominant isoform in nuclear extracts from GH-treated UMR 106 cells.

The GH secretory pattern in adult male rats is characterized by plasma GH pulses of 1-h duration every 3.5-4 h, with a typical peak plasma GH concentration of 200 ng/ml, while females exhibit more continuous plasma GH levels of about 20–40 ng/ml.(29) Pulsatile GH is more effective than continuous GH in promoting weight gain and longitudinal bone growth. The mechanism by which a temporal plasma profile of GH regulates this physiological response is not well understood. The STAT5 gene deletion experiments described previously showed that lack of STAT5B in male mice resulted in reduced body growth. From our experiments, it can be concluded that stimulation of osteoblast cells with one pulse of GH, with a concentration seen in males, activates both STAT5A and STAT5B. This same concentration also induced similar nuclear entry and nuclear disappearance of STAT5A and STAT5B protein. Thus, it is not likely that selective activation of STAT5 isoforms could explain the effect of sex-differentiated GH secretory patterns on bone growth. In male mice with both STAT5A and STAT5B genes deleted body growth was reduced further. Furthermore, in female mice with both STAT5 genes deleted body growth was also reduced. This is an indication that both STAT5 isoforms probably participate in regulating genes important for growth. Further studies are needed to identify these genes and to describe the role of each STAT5 isoform for activation of these genes.

To obtain maximal transcriptional activity, some STATs require serine phosphorylation, in addition to tyrosine phosphorylation.(31) In the case of STAT5A, a recent study has shown GH-induced activation of mitogen-activated protein kinase (MAPK), which results in phosphorylation of a serine residue in the C-terminal activation domain of STAT5A.(32) STAT5B also has been shown to be serine phosphorylated after stimulation of cells with PRL.(22,33) In our time-course study (Fig. 2), both STAT5A and STAT5B had a lower electrophoretic mobility after 30–60 minutes of treatment of UMR 106 cells with GH. This experimental finding could be an indication of GH-induced serine phosphorylation of STAT5A and STAT5B in UMR 106 cells. Further studies are needed to resolve this issue.

Both STAT5A and STAT5B proteins were shown to be present in nuclear extracts after GH stimulation of UMR 106 cells. To determine if both isoforms displayed DNA-binding activity and if formation of homodimers and heterodimers and their relative levels could be determined, supershift experiments with antibodies recognizing both isoforms or each isoform individually were performed (Fig. 4). Addition of these antibodies resulted in each case in two supershifted complexes. The complexes supershifted with isoform-specific antibodies had slightly different electrophoretic mobility as compared with the complexes supershifted with STAT5 antibody. This experiment clearly showed that both isoforms were activated to DNA-binding form by GH. The appearance of two supershifted complexes with each antibody could be an indication of STAT5A and STAT5B homo- and heterodimers with different electrophoretic mobility. It cannot be excluded that faster and slower migrating DNA-protein complexes could have been caused by binding of one and two antibodies, respectively. Thus from these data it is not possible to determine the presence of STAT5A and STAT5B heterodimers or the levels of homodimers and heterodimers in nuclear extracts from GH-treated UMR 106 cells. More experiments and other tools are needed to do this.

In conclusion, we have shown that the rat osteoblast-like osteosarcoma cell line UMR 106 expresses functional GH-JAK2/STAT5 signaling pathway. Furthermore, these cells express both isoforms of STAT5. The recent finding of the importance of STAT5 for body growth together with the fact that, at present, limited knowledge exists regarding intracellular pathways and genes activated directly by GH in osteoblasts calls for intensified research. To resolve these issues, different experimental models are needed. In this study, we have shown that the osteoblast-like osteosarcoma cell line UMR 106 is a suitable experimental model to study unique GH effects in osteoblasts mediated by STAT5.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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