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Rab7b, a novel lysosome-associated small GTPase, is involved in monocytic differentiation of human acute promyelocytic leukemia cells
BBRC Biochemical and Biophysical Research Communications 318 (2004) 792–799 www.elsevier.com/locate/ybbrc
Rab7b, a novel lysosome-associated small GTPase, is involved in monocytic differentiation of human acute promyelocytic leukemia cells Mingjin Yang,a,1 Taoyong Chen,b,1 Chaofeng Han,a Nan Li,b Tao Wan,b and Xuetao Caoa,b,* b
a Institute of Immunology, Zhejiang University, 353 Yanan Road, Hangzhou 310031, PR China Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China
Received 27 March 2004 Available online
Abstract Rab7 is a small Rab GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Here we report the cloning and characterization of a novel Rab7-like GTPase, which shares highest homology with Rab7 and thus is designated as Rab7b. Northern blot analysis shows that Rab7b mRNA is expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte. RT-PCR or Western blot analysis of Rab7b expression shows that Rab7b is selectively expressed in monocytes, monocyte-derived immature dendritic cells (DCs), and promyeloid or monocytic leukemia cell lines. In the peripheral blood, Rab7b is specifically detected in CD14þ cells, but not in CD4þ , CD8þ , CD19þ or CD56þ cells. When immature DCs are matured with lipopolysaccharide (LPS), Rab7b expression is gradually downregulated, while Rab7b is upregulated when monocytes are activated by LPS treatments. In acute promyelocytic leukemia (APL) HL-60 and NB4 cell lines, Rab7b expression is upregulated after phorbol myristate acetate (PMA)-induced monocytic differentiation. By immunofluorescence confocal microscopy, we demonstrate that Rab7b is associated with lysosomal organelles. Our data suggest that Rab7b is a lysosome-localized monocytic cellspecific small GTPase, and is involved in PMA-induced APL cell differentiation and possibly in regulation of monocyte functions. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Gene expression; Rab7; Monocyte; Dendritic cell; Myeloid cell; Acute promyelocytic leukemia; Lysosome
Rab proteins are small GTPases consisting of more than 40 members, and belonged to the Ras superfamily that specifically bind GTP/GDP and display an endogenous GTPase activity [1–3]. Rab proteins are involved in various aspects of endocytic and exocytic protein transport by specific association with membrane vesicles or organelles, such as endosomes, secretory vesicles, lysosomes, Golgi apparatus, and plasma membranes [1– 3]. In its GTP-binding active form, Rab proteins are membrane-associated; however, in its GDP-binding inactive form, they are mainly distributed in plasma. The GTP/GDP switch of Rab proteins is dependent on the *
Corresponding author. Fax: +86-571-8721-7410. E-mail address: [email protected] (X. Cao). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.115
endogenous GTPase activity, which is regulated by additional factors, such as GDP exchange factor and GTPase-activating protein [1–3]. Rab7 is one of the Rab GTPases that regulates vesicular traffic from early to late endosomal stages of endocytic pathway [4]. Rab7 of human, mouse, rat, and plant origin have been identified [5–8], showing that Rab7 is widely distributed and involved in multiple protein transport events. The Rab7-regulated events include phagosome maturation, Trypanosoma cruzi invasion, osteoblast polarization, early-late endosome fusion, and maturation of Salmonella typhimuriumcontaining vacuoles [9–13]. Recently, it is reported that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 may be involved in the process of atherogenesis [14,15]. Rab7 plays important roles in major
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histocompatibility class II (MHC-II)-restricted antigen presentation in B cells, and regulates the rate of antigen presentation [16]. Therefore, identification of novel Rab7 homologues will be helpful in elucidation of Rab7 function. In the previous studies, we discovered some novel genes from a cDNA library of human peripheral monocyte-derived dendritic cells (MoDCs) [17–19]. In the present study, we describe the functional characterization of a novel lysosomal Rab GTPase that shares more than approximately 68% similarity with Rab7, thus designated as Rab7b. Our study suggests that Rab7b is selectively expressed in monocytic cells, and involved in the phorbol myristate acetate (PMA)-induced monocytic differentiation of acute promyelocytic leukemia (APL).
Materials and methods Cell preparation, cell culture, and cell treatment. Human MoDCs were cultured as described previously [20]. Cells from peripheral blood specifically expressing CD14, CD56, CD4, CD8 or CD19 were isolated with immunomagnetic beads (Miltenyi Biotech, Bergische Aladbach, Germany) as described [20,21]. Human monocytes were isolated from peripheral blood as described [17,20]. Acute monocytic leukemia THP1 (TIB-202), histiocytic lymphoma U-937 (CRL-1593), APL HL-60 (CCL-240), and chronic myelogenous leukemia K562 (CCL-243) were all obtained from ATCC and cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). NB4 (APL) was a kindly gift from Professor Guanlin Sun (Shanghai Second Medical University, Shanghai). For differentiation induction, 50 ng/ml PMA (Sigma) was supplemented into the culture medium and incubated for 4, 8, 12, 24, and 48 h, respectively. Then cells were collected and subjected to RNA isolation and cell lysate preparation. Identification of Rab7b full-length cDNA. Rab7b cDNA was directly isolated from the human dendritic cell (DC) cDNA-library by large-scale random sequencing [17]. By comparison with gene databases, we identified Rab7b as a novel Rab GTPase. The cDNA sequence of Rab7b was confirmed by sequencing of the RT-PCR product from MoDC by using specific primers 50 GGGAAGTGG CGTGGCTTC 30 and 50 CAGAGCAGGCGTCTGGAG 30 . Homologous alignment and chromosomal localization were performed with the software provided by NCBI database. Northern blotting. Northern blotting was performed as described previously [17,22]. The ready-to-use human Northern blot filters containing 2 lg/lane mRNA were purchased from Clontech Laboratories (Palo Alto, CA) and hybridized with [32 p]dCTP-labeled full-length Rab7b probe as described [17,18]. Otherwise, total RNA (10 lg/lane) was isolated from leukemia cell lines as described [18,23], separated on 1% agarose, transferred to Hybond Nþ nylon transfer membrane (Amersham–Pharmacia Biotech.), and hybridized with the above probe. The 18S RNA separated on 1% ethidium bromide (EB)-positive agarose was taken as RNA quantification control. RT-PCR analysis. Total cellular RNA isolation and RT-PCR assay were performed as described previously [22,23]. Primers for human Rab7b were 50 TACAGATCTGGGACACGG 30 (sense) and 50 GTC AGCACGATCTGCTCC 30 (anti-sense), and the expected product about 429 bp. Primers for human b-actin were 50 -GCATCGTGA TGGACTCCG-30 (sense) and 50 -TCGGAAGGTGGACAGCGA-30 (antisense). Recombinant expression of GST-Rab7b protein and polyclonal antibody preparation. For expression of recombinant GST-Rab7b fusion
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protein, full-length Rab7b cDNA containing BamHI/EcoRI sites was cloned into pGEX-2T vector (Amersham–Pharmacia Biotech.). Escherichia coli strain BL21 was used as a host. Purification of GST-Rab7b fusion protein, immunization of rabbits, and preparation of polyclonal antibody serum were performed as described [18,19]. For further purification of the polyclonal antibody, serum was chromatographed through the GST-expressing E. coli, lysate-embedded columns (Pierce) and then purified by protein-A chromatography. Cellular lysate preparation and Western blot analysis. Cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, and 1 mM Na3 VO4 plus 1 mM PMSF. Then brief sonication was performed on ice. Lysates were collected by centrifugation at 4 °C for 10 min at 10,000g. Protein concentration was determined by BCA Protein assay kit as instructed (Pierce). Western blot assay was performed as described [23]. Expression vector construction and gene transfection. The construction of eukaryotic expression vectors encoding green fluorescence protein (GFP)-Rab7b or Rab7b was performed as described [18,19]. For transient gene expression in HL-60 cells, Effectene Transfection Reagent was used according to protocols recommended (Qiagen). Immunofluorescence confocal microscopy. Transiently transfected HL-60 cells expressing GFP-Rab7b or GFP were cultured on polylysine-treated coverslips, stained with 100 nM LycoTracker Red (578/599, Molecular Probes, Eugene, OR) for 15 min at 37 °C, fixed in 4% polyformaldehyde for 15 min, and observed by fluorescence confocal microscopy (LSM Confocal Microscope, Carl Zeiss). Alternatively, HL-60 cells cultured on coverslips, with or without PMA (50 ng/ml) treatments, were incubated sequentially with Rab7b polyclonal Ab (dilution 1:50) and Oregon-green 488-conjugated antiIgG antibody (Molecular probes, dilution 1:100). Finally, the cells were rinsed in PBS and viewed under a fluorescence confocal microscope. FACS assay. HL-60 cells treated with or without PMA (50 ng/ml) were labeled with FITC-conjugated anti-CD11b, -CD14 or -CD33 Ab (BD Pharmingen) as described previously [20,23]. For indirect fluorescent labeling of Rab7b, HL-60 cells treated as above were permeabilized with 0.05% saponin in PBS, and sequentially labeled with Rab7b Ab and FITC-labeled anti-rabbit IgG in the presence of 10% FCS and 2 mg/ml bovine serum albumin (BSA) at 4 °C. Normal rabbit IgG and corresponding isotype controls (BD PharMingen) were used as non-specific controls.
Results Identification of Rab7b The cDNA sequence encoding Rab7b was directly isolated from DC cDNA library by large-scale sequencing. The cDNA is of 1571 bp, contains a complete open reading fame (ORF) of 600 bp, which potentially encodes a 199-residue protein, and is deposited in GenBank under Accession No. AY094596. Upstream the start codon (ATG), we found an in-frame stop codon (TGA, )12), indicating that the cDNA encoding Rab7b was of full-length. Rab7b protein contains three protein kinase c (PKC) phosphorylation sites (56–58, 153–155, 186–188 aa), three casein kinase II (CKII) phosphorylation sites (32–35, 91–94, 177–180 aa), one tyrosine kinase site (143–149 aa), three N-myristoylation sites (18–23, 80–85, 83–88 aa), one cell attachment sequence (103–105 aa), and one ATP/GTP binding site
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motif (15–22 aa), as predicted by ScanProsite assay. Additionally, a –Cys–Cys– motif, which has been shown to be important for post-translational modification and crucial for targeting of Rab proteins to specific cellular compartments [24–27], is found at the carboxyl terminus (Fig. 1A). Homology analysis was performed using BLAST program. At nucleic acid level, Rab7b shares highest homology with human BC017092 (99%) and mouse BC019395 (85%). At protein level, it shares highest homology with AAH17092 and was similar to AAH19395 (89%). Rab7b shows more than 50% identity and 65% similarity with human Rab7 proteins, thus designated as Rab7b. A murine clone (NM_145590) also encodes a 199-residue protein and shares 92% similarity with human Rab7b protein, which may be the mouse
homologue of human Rab7b (Figs. 1A and B). By Blasting genome database, we found that human and mouse Rab7b were overlapping with UniGene Cluster Hs. 55405 (1q32) and Mm.44508 (1E4), respectively, while Rab7 was mapped to 3q21.3 in human and 6C36D1 in mouse. Generally, Rab7 and Rab7-like proteins could be classified into two classes, based on the carboxyl terminal –C–C– or –C–X–C– motif, where “C” indicates cysteine and “X” any amino acid. The –C–C– motifcontaining members include AY094596 of Homo sapiens, NM_145509 of Mus musculus, AY050242 of Paramecium aurelia, AJ290938 of Plasmodium falciparum, and AY226827 of Oryza sativa, while the –C–X–C– motifcontaining members include NM_200928 of Danio rerio, NM_064148 of Caenorhabditis elegans, NM_079748 of
Fig. 1. Bioinformatic analysis of Rab7b and Rab7 proteins. (A) Multiple alignment of Rab7b with closely related Rab family members. Alignment was performed with the GCG package and minimally adjusted manually. Identical residues are boxed in black and similar residues are in gray. Conserved residues involved in GTP-binding (black triangle), GTPase activity (black star), and C-terminal –C–C– motif (black dot) were labeled. (B,C) Phylogenetic tree showing relationship of Rab7b to closely related Rab7 proteins. In (C), arrows with dotted line indicated for proteins with –C–C– motif, and arrows with solid line for members with –C–X–C– motif.
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Drosophila melanogaster, BC060401 of Xenopus laevis, AB072431 of Aspergillus nidulans, NM_009005 of M. musculus, NM_023950 of Rattus norvegicus, and U44104 of H. sapiens in the currently available databases (Fig. 1C). However, the 199-residue Rab7b proteins are only found in human and mouse, while Rab7 proteins are conventional among various species (Fig. 1C). Therefore, Rab7b is a homologue of Rab7 and possibly functions deferentially to Rab7. Expression pattern of Rab7b By Northern blot analysis, we found that Rab7b was expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte (PBL), and that the single transcript was about 2.5 kb (Fig. 2A). By RTPCR analysis of both hematological cell lines and solid tumor cell lines, we found that Rab7b mRNA was specifically expressed in promyeloid or monocytic leukemia cell lines (Figs. 2B and C). To examine the
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specificity of the prepared polyclonal Ab, we transiently transfected Rab7b-negative HeLa cells with plasmid encoding full-length Rab7b, and found that the polyclonal Ab recognized a protein band of about 26 kDa, corresponding to the calculated molecular weight for Rab7, both in HeLa-Rab7b cells and in promyeloid and monocytic leukemia cell lines (Fig. 2C). In murine cell lines, Rab7b mRNA was detected in NIH3T3 and Raw 264.7 cells, but not in EL-4 and P815, 3LL, and B16 cells (Fig. 2D). In murine tissues, Rab7b mRNA was detected in brain, lung, skeletal muscle, colon, ovary, and testis, but not in heart, kidney, liver, spleen, small intestine, and thymus (Fig. 2E). Specific expression of Rab7b in monocytic cells and monocyte-derived immature DC To further examine the expression pattern of Rab7b, we isolated CD4þ , CD8þ , CD56þ , CD14þ , and CD19þ cells from peripheral blood of healthy adult volunteers,
Fig. 2. Expression pattern of Rab7b in tissues and cell lines. (A) Northern blot assay of human Rab7b expression. The Northern blot filters containing 2 lg/lane mRNA were probed with full-length Rab7b cDNA. (B,C) Expression of human Rab7b in human cell lines. (D,E) RT-PCR assay of mouse Rab7b expression in cell lines (D) and tissues (E). b-Actin was used as controls. WB, Western blot.
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Fig. 3. Analysis of Rab7b expression in monocyte-derived cells. (A) Examination of Rab7b expression in subpopulations of peripheral blood cells. Subpopulations of cells were isolated and subjected to both RT-PCR and Western blot (WB) assay. (B) Expression of Rab7b in human monocytes and DCs. Monocytes and DCs derived from human peripheral monocytes were subjected to LPS (100 ng/ml) stimulation as indicated and examined for Rab7b expression. Equal amounts of protein (30 lg/lane) were loaded. b-Actin was used as controls. WB, Western blot.
or prepared peripheral blood monocyte-derived DCs. We found that Rab7b was specifically expressed in monocytic cells (CD14þ cells) and in immature DC, but not in CD4þ /CD8þ T cells, CD19þ B cells, CD56þ natural killer cells (NK) or LPS-matured DC, as detected by RT-PCR and Western blot assay (Figs. 3A and B). In human monocytes, Rab7b was increased steadily upon LPS stimulation, indicating that Rab7b may be involved in functions of activated monocytes (Fig. 3B). Association of Rab7b with monocytic differentiation of acute promyelocytic myeloid leukemia As Rab7b was specifically expressed in promyeloid and monocytic leukemia cell lines, we then investigated whether Rab7b was associated with monocytic APL differentiation. We stimulated APL HL-60 and NB4 cells with 50 ng/ml PMA for 4, 8, 12, 24, and 48 h, respectively. Then we collected the cells and performed RT-PCR and Northern blot assay. We found that Rab7b mRNA was significantly upregulated both in HL-60 (Fig. 4A) and NB4 cells (Fig. 4B) after PMA treatments. After PMA treatment of HL-60 cells and NB4 cells, Rab7b mRNA was present or increased as early as 4 h and reached maximum at 12 h (Figs. 4A and B). As controls, we also stimulated Molt-4 T lymphoma and Raji B lymphoma with PMA, performed RT-PCR, and found that Rab7b was not regulated by
Fig. 4. Upregulation of Rab7b in the differentiation of acute promyelocytic leukemia HL-60 and NB4 cell lines. HL-60 (A) and NB4 cells (B) were treated with PMA (50 ng/ml) as indicated and collected at indicated time points. RT-PCR and Northern blot were used to examine Rab7b expression. b-Actin was used as control in RT-PCR. In Northern blot assay, total RNA was separated on EB-positive 1% agarose and the 18S RNA was used as loading control. [32 p]dCTPlabeled full-length Rab7b cDNA was applied as probe. (C) FACS analysis of differentiation markers of HL-60 cells treated with PMA (50 ng/ml) for 24 h. Weak dark lines indicated for isotypic Ab or polyclonal Ab controls, and dark lines for specific Abs as indicated. Numbers above each panel indicated for mean fluorescence intensity. Corresponding isotype control Ab and normal rabbit IgG were used as controls.
PMA and remained negative during the treatments (data not shown). It has been reported that HL-60 and NB4 cells can differentiate into monocytic cells after PMA treatment,
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leading to increased expression of CD14 and CD11b, and decreased expression of CD33 [27–32]. We therefore examined the expression of CD14, CD11b, CD33, and Rab7b by FACS assay in HL-60 cells treated with PMA (50 ng/ml) for 24 h. We found that after PMA stimulation, expression of intracellular Rab7b was greatly increased, concomitant with the upregulation of CD11b and CD14 and the downregulation of CD33, indicating that Rab7b expression is associated with monocytic differentiation of HL-60 cells (Fig. 4C). Similar results were found for NB4 cells (data not shown). Lysosomal localization of Rab7b HL-60 cells cultured on poly-lysine-treated coverslips were transiently transfected with GFP-Rab7b expression vector, stained with LysoTracker Red, a lysosomespecific pH-dependent fluorescent dye, and viewed under confocal microscope. We found that Rab7b was colocalized with LysoTracker (Fig. 5B) while GFP alone was localized in the nucleus (Fig. 5A), suggesting that Rab7b was a lysosome-associated protein. Additionally, we found that Rab7b was not colocalized with mitochondria and Golgi dyes (data not shown). As further examination, HL-60 cells treated with PMA (50 ng/ml) for 24 h were stained with Rab7b polyclonal Ab for detection of endogenous Rab7b. Endogenous Rab7b
Fig. 5. Examination of Rab7b subcellular localization by immunofluorescence confocal microscopy. HL-60 cells transiently transfected with GFP (A) or GFP-Rab7b (B) expression vectors, stained with Lysotracker Red. Otherwise, normal HL-60 cells (C) and HL-60 cells treated with PMA (50 ng/ml, D) for 24 h were labeled with Rab7b Ab and Oregon-green 488 secondary Ab. Cells were viewed under confocal microscope. Bar ¼ 50 lm.
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was also colocalized with LysoTracker Red (Fig. 5D) while untreated HL-60 cells showed no positive staining (Fig. 5C).
Discussion In the present study, we have cloned and characterized a novel Rab7 homologue, Rab7b, which is selectively expressed in promyeloid or monocytic leukemia cell lines, and specifically expressed in normal monocytes and monocyte-derived immature DC in peripheral blood. Therefore, Rab7b is expressed in a rather specific pattern and can potentially serve as a new monocytic cell marker. It is shown that phosphoinositide 3-kinase (PI3K), protein kinase B/Akt, and Rab7 were sequentially activated during macropinosome formation [33]. Since conserved motifs involved in PKC and tyrosine kinase phosphorylation, or GTP-binding and -hydrolysis, are found in Rab7b, we predict here that Rab7b is presumably a Rab GTPase that can specifically bind GTP/ GDP and hydrolyze GTP, and possibly regulates protein transport to or from lysosomes under the regulation of kinases, such as PKC and PI3K. Rab proteins are important regulators of protein transport along the endocytic and exocytic pathways [1– 3]. Rab7 has been implicated in the regulation of protein transport from early to late endosomes and it has been used as a late lysosome marker [4–13]. During endocytosis of fluid phase marker horseradish peroxidase (HRP), Rab7 functions in a sequential manner downstream of Rab5, which is associated with early endosomes and involved in the regulation of early endocytosis [34,35]. As Rab7 has been suggested to be associated with controlling of aggregation and fusion of late endocytic structures/lysosomes, it has been implicated in the degradation of proteins and maintenance of the perinuclear lysosome compartment [36]. High homology has been found between Rab7b and Rab7, and Rab7b is associated with lysosomes or acidic pH compartments. Thus, Rab7b may function similar to Rab7, that is, protein transport to or from lysosomes. However, Rab7b possibly only serves as regulator of protein transport in certain cell types or under special conditions, considering that Rab7b is preferentially expressed in DC and CD14þ monocytic cells. DCs and monocytes are potent antigen-presenting cells that can uptake and process exogenous antigens, and present antigens to T cells [37,38]. It is shown that Rab7 plays a role in MHC II molecule-associated antigen presentation [16]. Thus, it is possible that Rab7b is possibly involved in endocytosis and presentation of protein antigens. The roles of Rab7b in antigen uptake and presentation by antigenpresenting cells, such as DC and monocytes/macrophages, are now undergoing.
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Acute promyelocytic leukemia HL-60 and NB4 cells have been used to study leukemia differentiation. After stimulation with PMA, they differentiate mainly towards monocytic lineage, concomitant with adhesion to support matrix, increased phagocytosis capacity, restored respiratory burst, impaired growth rate, and inhibited tumorigenicity [27–32]. Rab7b expression is gradually increased in HL-60 and NB4 cells upon PMA treatments, together with alterations in monocytic differentiation markers, such as upregulation of CD11b and CD14, and downregulation of CD33. It has been demonstrated previously that several Rab proteins, including Rab7, Rab2, and Rab11b, are transcriptionally increased upon PMA treatments [39]. But it remains obscure whether the upregulation of Rab proteins expression is the causing effects of PMA treatments, or the Rab proteins are the effectors that work together to facilitate the restore of the normal functions of leukemia cells. In case of Rab7b upregulation in myeloid leukemia cells after PMA stimulation, a reasonable explanation is that PMA increases Rab7b expression, which in turn promotes the intermediate or late phase differentiation of leukemia cells and restores the function of myeloid cells, such as restored phagocytosis, respiratory burst, and antigen presentation capacity. The mechanisms and the functions of Rab7b in leukemia cell differentiation need further study. However, our studies at least suggest that Rab7b, a novel lysosomal Rab7-like protein, is associated with monocytic differentiation of APL cells.
Acknowledgments This work was supported by grants from the National Key Basic Research Program of China (2001CB510002), the National Natural Science Foundation of China (30121002), and the National High Biotechnology Development Program of China (2002BA711A01). We sincerely thank Qiangsu Guo, Mei Jin, Yan Li, Xianwei Ma, and Xiaoting Zuo for their excellent technical assistance.
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Rab7b, a novel lysosome-associated small GTPase, is involved in monocytic differentiation of human acute promyelocytic leukemia cells Mingjin Yang,a,1 Taoyong Chen,b,1 Chaofeng Han,a Nan Li,b Tao Wan,b and Xuetao Caoa,b,* b
a Institute of Immunology, Zhejiang University, 353 Yanan Road, Hangzhou 310031, PR China Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China
Received 27 March 2004 Available online
Abstract Rab7 is a small Rab GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Here we report the cloning and characterization of a novel Rab7-like GTPase, which shares highest homology with Rab7 and thus is designated as Rab7b. Northern blot analysis shows that Rab7b mRNA is expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte. RT-PCR or Western blot analysis of Rab7b expression shows that Rab7b is selectively expressed in monocytes, monocyte-derived immature dendritic cells (DCs), and promyeloid or monocytic leukemia cell lines. In the peripheral blood, Rab7b is specifically detected in CD14þ cells, but not in CD4þ , CD8þ , CD19þ or CD56þ cells. When immature DCs are matured with lipopolysaccharide (LPS), Rab7b expression is gradually downregulated, while Rab7b is upregulated when monocytes are activated by LPS treatments. In acute promyelocytic leukemia (APL) HL-60 and NB4 cell lines, Rab7b expression is upregulated after phorbol myristate acetate (PMA)-induced monocytic differentiation. By immunofluorescence confocal microscopy, we demonstrate that Rab7b is associated with lysosomal organelles. Our data suggest that Rab7b is a lysosome-localized monocytic cellspecific small GTPase, and is involved in PMA-induced APL cell differentiation and possibly in regulation of monocyte functions. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Gene expression; Rab7; Monocyte; Dendritic cell; Myeloid cell; Acute promyelocytic leukemia; Lysosome
Rab proteins are small GTPases consisting of more than 40 members, and belonged to the Ras superfamily that specifically bind GTP/GDP and display an endogenous GTPase activity [1–3]. Rab proteins are involved in various aspects of endocytic and exocytic protein transport by specific association with membrane vesicles or organelles, such as endosomes, secretory vesicles, lysosomes, Golgi apparatus, and plasma membranes [1– 3]. In its GTP-binding active form, Rab proteins are membrane-associated; however, in its GDP-binding inactive form, they are mainly distributed in plasma. The GTP/GDP switch of Rab proteins is dependent on the *
Corresponding author. Fax: +86-571-8721-7410. E-mail address: [email protected] (X. Cao). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.115
endogenous GTPase activity, which is regulated by additional factors, such as GDP exchange factor and GTPase-activating protein [1–3]. Rab7 is one of the Rab GTPases that regulates vesicular traffic from early to late endosomal stages of endocytic pathway [4]. Rab7 of human, mouse, rat, and plant origin have been identified [5–8], showing that Rab7 is widely distributed and involved in multiple protein transport events. The Rab7-regulated events include phagosome maturation, Trypanosoma cruzi invasion, osteoblast polarization, early-late endosome fusion, and maturation of Salmonella typhimuriumcontaining vacuoles [9–13]. Recently, it is reported that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 may be involved in the process of atherogenesis [14,15]. Rab7 plays important roles in major
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histocompatibility class II (MHC-II)-restricted antigen presentation in B cells, and regulates the rate of antigen presentation [16]. Therefore, identification of novel Rab7 homologues will be helpful in elucidation of Rab7 function. In the previous studies, we discovered some novel genes from a cDNA library of human peripheral monocyte-derived dendritic cells (MoDCs) [17–19]. In the present study, we describe the functional characterization of a novel lysosomal Rab GTPase that shares more than approximately 68% similarity with Rab7, thus designated as Rab7b. Our study suggests that Rab7b is selectively expressed in monocytic cells, and involved in the phorbol myristate acetate (PMA)-induced monocytic differentiation of acute promyelocytic leukemia (APL).
Materials and methods Cell preparation, cell culture, and cell treatment. Human MoDCs were cultured as described previously [20]. Cells from peripheral blood specifically expressing CD14, CD56, CD4, CD8 or CD19 were isolated with immunomagnetic beads (Miltenyi Biotech, Bergische Aladbach, Germany) as described [20,21]. Human monocytes were isolated from peripheral blood as described [17,20]. Acute monocytic leukemia THP1 (TIB-202), histiocytic lymphoma U-937 (CRL-1593), APL HL-60 (CCL-240), and chronic myelogenous leukemia K562 (CCL-243) were all obtained from ATCC and cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). NB4 (APL) was a kindly gift from Professor Guanlin Sun (Shanghai Second Medical University, Shanghai). For differentiation induction, 50 ng/ml PMA (Sigma) was supplemented into the culture medium and incubated for 4, 8, 12, 24, and 48 h, respectively. Then cells were collected and subjected to RNA isolation and cell lysate preparation. Identification of Rab7b full-length cDNA. Rab7b cDNA was directly isolated from the human dendritic cell (DC) cDNA-library by large-scale random sequencing [17]. By comparison with gene databases, we identified Rab7b as a novel Rab GTPase. The cDNA sequence of Rab7b was confirmed by sequencing of the RT-PCR product from MoDC by using specific primers 50 GGGAAGTGG CGTGGCTTC 30 and 50 CAGAGCAGGCGTCTGGAG 30 . Homologous alignment and chromosomal localization were performed with the software provided by NCBI database. Northern blotting. Northern blotting was performed as described previously [17,22]. The ready-to-use human Northern blot filters containing 2 lg/lane mRNA were purchased from Clontech Laboratories (Palo Alto, CA) and hybridized with [32 p]dCTP-labeled full-length Rab7b probe as described [17,18]. Otherwise, total RNA (10 lg/lane) was isolated from leukemia cell lines as described [18,23], separated on 1% agarose, transferred to Hybond Nþ nylon transfer membrane (Amersham–Pharmacia Biotech.), and hybridized with the above probe. The 18S RNA separated on 1% ethidium bromide (EB)-positive agarose was taken as RNA quantification control. RT-PCR analysis. Total cellular RNA isolation and RT-PCR assay were performed as described previously [22,23]. Primers for human Rab7b were 50 TACAGATCTGGGACACGG 30 (sense) and 50 GTC AGCACGATCTGCTCC 30 (anti-sense), and the expected product about 429 bp. Primers for human b-actin were 50 -GCATCGTGA TGGACTCCG-30 (sense) and 50 -TCGGAAGGTGGACAGCGA-30 (antisense). Recombinant expression of GST-Rab7b protein and polyclonal antibody preparation. For expression of recombinant GST-Rab7b fusion
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protein, full-length Rab7b cDNA containing BamHI/EcoRI sites was cloned into pGEX-2T vector (Amersham–Pharmacia Biotech.). Escherichia coli strain BL21 was used as a host. Purification of GST-Rab7b fusion protein, immunization of rabbits, and preparation of polyclonal antibody serum were performed as described [18,19]. For further purification of the polyclonal antibody, serum was chromatographed through the GST-expressing E. coli, lysate-embedded columns (Pierce) and then purified by protein-A chromatography. Cellular lysate preparation and Western blot analysis. Cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, and 1 mM Na3 VO4 plus 1 mM PMSF. Then brief sonication was performed on ice. Lysates were collected by centrifugation at 4 °C for 10 min at 10,000g. Protein concentration was determined by BCA Protein assay kit as instructed (Pierce). Western blot assay was performed as described [23]. Expression vector construction and gene transfection. The construction of eukaryotic expression vectors encoding green fluorescence protein (GFP)-Rab7b or Rab7b was performed as described [18,19]. For transient gene expression in HL-60 cells, Effectene Transfection Reagent was used according to protocols recommended (Qiagen). Immunofluorescence confocal microscopy. Transiently transfected HL-60 cells expressing GFP-Rab7b or GFP were cultured on polylysine-treated coverslips, stained with 100 nM LycoTracker Red (578/599, Molecular Probes, Eugene, OR) for 15 min at 37 °C, fixed in 4% polyformaldehyde for 15 min, and observed by fluorescence confocal microscopy (LSM Confocal Microscope, Carl Zeiss). Alternatively, HL-60 cells cultured on coverslips, with or without PMA (50 ng/ml) treatments, were incubated sequentially with Rab7b polyclonal Ab (dilution 1:50) and Oregon-green 488-conjugated antiIgG antibody (Molecular probes, dilution 1:100). Finally, the cells were rinsed in PBS and viewed under a fluorescence confocal microscope. FACS assay. HL-60 cells treated with or without PMA (50 ng/ml) were labeled with FITC-conjugated anti-CD11b, -CD14 or -CD33 Ab (BD Pharmingen) as described previously [20,23]. For indirect fluorescent labeling of Rab7b, HL-60 cells treated as above were permeabilized with 0.05% saponin in PBS, and sequentially labeled with Rab7b Ab and FITC-labeled anti-rabbit IgG in the presence of 10% FCS and 2 mg/ml bovine serum albumin (BSA) at 4 °C. Normal rabbit IgG and corresponding isotype controls (BD PharMingen) were used as non-specific controls.
Results Identification of Rab7b The cDNA sequence encoding Rab7b was directly isolated from DC cDNA library by large-scale sequencing. The cDNA is of 1571 bp, contains a complete open reading fame (ORF) of 600 bp, which potentially encodes a 199-residue protein, and is deposited in GenBank under Accession No. AY094596. Upstream the start codon (ATG), we found an in-frame stop codon (TGA, )12), indicating that the cDNA encoding Rab7b was of full-length. Rab7b protein contains three protein kinase c (PKC) phosphorylation sites (56–58, 153–155, 186–188 aa), three casein kinase II (CKII) phosphorylation sites (32–35, 91–94, 177–180 aa), one tyrosine kinase site (143–149 aa), three N-myristoylation sites (18–23, 80–85, 83–88 aa), one cell attachment sequence (103–105 aa), and one ATP/GTP binding site
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motif (15–22 aa), as predicted by ScanProsite assay. Additionally, a –Cys–Cys– motif, which has been shown to be important for post-translational modification and crucial for targeting of Rab proteins to specific cellular compartments [24–27], is found at the carboxyl terminus (Fig. 1A). Homology analysis was performed using BLAST program. At nucleic acid level, Rab7b shares highest homology with human BC017092 (99%) and mouse BC019395 (85%). At protein level, it shares highest homology with AAH17092 and was similar to AAH19395 (89%). Rab7b shows more than 50% identity and 65% similarity with human Rab7 proteins, thus designated as Rab7b. A murine clone (NM_145590) also encodes a 199-residue protein and shares 92% similarity with human Rab7b protein, which may be the mouse
homologue of human Rab7b (Figs. 1A and B). By Blasting genome database, we found that human and mouse Rab7b were overlapping with UniGene Cluster Hs. 55405 (1q32) and Mm.44508 (1E4), respectively, while Rab7 was mapped to 3q21.3 in human and 6C36D1 in mouse. Generally, Rab7 and Rab7-like proteins could be classified into two classes, based on the carboxyl terminal –C–C– or –C–X–C– motif, where “C” indicates cysteine and “X” any amino acid. The –C–C– motifcontaining members include AY094596 of Homo sapiens, NM_145509 of Mus musculus, AY050242 of Paramecium aurelia, AJ290938 of Plasmodium falciparum, and AY226827 of Oryza sativa, while the –C–X–C– motifcontaining members include NM_200928 of Danio rerio, NM_064148 of Caenorhabditis elegans, NM_079748 of
Fig. 1. Bioinformatic analysis of Rab7b and Rab7 proteins. (A) Multiple alignment of Rab7b with closely related Rab family members. Alignment was performed with the GCG package and minimally adjusted manually. Identical residues are boxed in black and similar residues are in gray. Conserved residues involved in GTP-binding (black triangle), GTPase activity (black star), and C-terminal –C–C– motif (black dot) were labeled. (B,C) Phylogenetic tree showing relationship of Rab7b to closely related Rab7 proteins. In (C), arrows with dotted line indicated for proteins with –C–C– motif, and arrows with solid line for members with –C–X–C– motif.
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Drosophila melanogaster, BC060401 of Xenopus laevis, AB072431 of Aspergillus nidulans, NM_009005 of M. musculus, NM_023950 of Rattus norvegicus, and U44104 of H. sapiens in the currently available databases (Fig. 1C). However, the 199-residue Rab7b proteins are only found in human and mouse, while Rab7 proteins are conventional among various species (Fig. 1C). Therefore, Rab7b is a homologue of Rab7 and possibly functions deferentially to Rab7. Expression pattern of Rab7b By Northern blot analysis, we found that Rab7b was expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte (PBL), and that the single transcript was about 2.5 kb (Fig. 2A). By RTPCR analysis of both hematological cell lines and solid tumor cell lines, we found that Rab7b mRNA was specifically expressed in promyeloid or monocytic leukemia cell lines (Figs. 2B and C). To examine the
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specificity of the prepared polyclonal Ab, we transiently transfected Rab7b-negative HeLa cells with plasmid encoding full-length Rab7b, and found that the polyclonal Ab recognized a protein band of about 26 kDa, corresponding to the calculated molecular weight for Rab7, both in HeLa-Rab7b cells and in promyeloid and monocytic leukemia cell lines (Fig. 2C). In murine cell lines, Rab7b mRNA was detected in NIH3T3 and Raw 264.7 cells, but not in EL-4 and P815, 3LL, and B16 cells (Fig. 2D). In murine tissues, Rab7b mRNA was detected in brain, lung, skeletal muscle, colon, ovary, and testis, but not in heart, kidney, liver, spleen, small intestine, and thymus (Fig. 2E). Specific expression of Rab7b in monocytic cells and monocyte-derived immature DC To further examine the expression pattern of Rab7b, we isolated CD4þ , CD8þ , CD56þ , CD14þ , and CD19þ cells from peripheral blood of healthy adult volunteers,
Fig. 2. Expression pattern of Rab7b in tissues and cell lines. (A) Northern blot assay of human Rab7b expression. The Northern blot filters containing 2 lg/lane mRNA were probed with full-length Rab7b cDNA. (B,C) Expression of human Rab7b in human cell lines. (D,E) RT-PCR assay of mouse Rab7b expression in cell lines (D) and tissues (E). b-Actin was used as controls. WB, Western blot.
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Fig. 3. Analysis of Rab7b expression in monocyte-derived cells. (A) Examination of Rab7b expression in subpopulations of peripheral blood cells. Subpopulations of cells were isolated and subjected to both RT-PCR and Western blot (WB) assay. (B) Expression of Rab7b in human monocytes and DCs. Monocytes and DCs derived from human peripheral monocytes were subjected to LPS (100 ng/ml) stimulation as indicated and examined for Rab7b expression. Equal amounts of protein (30 lg/lane) were loaded. b-Actin was used as controls. WB, Western blot.
or prepared peripheral blood monocyte-derived DCs. We found that Rab7b was specifically expressed in monocytic cells (CD14þ cells) and in immature DC, but not in CD4þ /CD8þ T cells, CD19þ B cells, CD56þ natural killer cells (NK) or LPS-matured DC, as detected by RT-PCR and Western blot assay (Figs. 3A and B). In human monocytes, Rab7b was increased steadily upon LPS stimulation, indicating that Rab7b may be involved in functions of activated monocytes (Fig. 3B). Association of Rab7b with monocytic differentiation of acute promyelocytic myeloid leukemia As Rab7b was specifically expressed in promyeloid and monocytic leukemia cell lines, we then investigated whether Rab7b was associated with monocytic APL differentiation. We stimulated APL HL-60 and NB4 cells with 50 ng/ml PMA for 4, 8, 12, 24, and 48 h, respectively. Then we collected the cells and performed RT-PCR and Northern blot assay. We found that Rab7b mRNA was significantly upregulated both in HL-60 (Fig. 4A) and NB4 cells (Fig. 4B) after PMA treatments. After PMA treatment of HL-60 cells and NB4 cells, Rab7b mRNA was present or increased as early as 4 h and reached maximum at 12 h (Figs. 4A and B). As controls, we also stimulated Molt-4 T lymphoma and Raji B lymphoma with PMA, performed RT-PCR, and found that Rab7b was not regulated by
Fig. 4. Upregulation of Rab7b in the differentiation of acute promyelocytic leukemia HL-60 and NB4 cell lines. HL-60 (A) and NB4 cells (B) were treated with PMA (50 ng/ml) as indicated and collected at indicated time points. RT-PCR and Northern blot were used to examine Rab7b expression. b-Actin was used as control in RT-PCR. In Northern blot assay, total RNA was separated on EB-positive 1% agarose and the 18S RNA was used as loading control. [32 p]dCTPlabeled full-length Rab7b cDNA was applied as probe. (C) FACS analysis of differentiation markers of HL-60 cells treated with PMA (50 ng/ml) for 24 h. Weak dark lines indicated for isotypic Ab or polyclonal Ab controls, and dark lines for specific Abs as indicated. Numbers above each panel indicated for mean fluorescence intensity. Corresponding isotype control Ab and normal rabbit IgG were used as controls.
PMA and remained negative during the treatments (data not shown). It has been reported that HL-60 and NB4 cells can differentiate into monocytic cells after PMA treatment,
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leading to increased expression of CD14 and CD11b, and decreased expression of CD33 [27–32]. We therefore examined the expression of CD14, CD11b, CD33, and Rab7b by FACS assay in HL-60 cells treated with PMA (50 ng/ml) for 24 h. We found that after PMA stimulation, expression of intracellular Rab7b was greatly increased, concomitant with the upregulation of CD11b and CD14 and the downregulation of CD33, indicating that Rab7b expression is associated with monocytic differentiation of HL-60 cells (Fig. 4C). Similar results were found for NB4 cells (data not shown). Lysosomal localization of Rab7b HL-60 cells cultured on poly-lysine-treated coverslips were transiently transfected with GFP-Rab7b expression vector, stained with LysoTracker Red, a lysosomespecific pH-dependent fluorescent dye, and viewed under confocal microscope. We found that Rab7b was colocalized with LysoTracker (Fig. 5B) while GFP alone was localized in the nucleus (Fig. 5A), suggesting that Rab7b was a lysosome-associated protein. Additionally, we found that Rab7b was not colocalized with mitochondria and Golgi dyes (data not shown). As further examination, HL-60 cells treated with PMA (50 ng/ml) for 24 h were stained with Rab7b polyclonal Ab for detection of endogenous Rab7b. Endogenous Rab7b
Fig. 5. Examination of Rab7b subcellular localization by immunofluorescence confocal microscopy. HL-60 cells transiently transfected with GFP (A) or GFP-Rab7b (B) expression vectors, stained with Lysotracker Red. Otherwise, normal HL-60 cells (C) and HL-60 cells treated with PMA (50 ng/ml, D) for 24 h were labeled with Rab7b Ab and Oregon-green 488 secondary Ab. Cells were viewed under confocal microscope. Bar ¼ 50 lm.
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was also colocalized with LysoTracker Red (Fig. 5D) while untreated HL-60 cells showed no positive staining (Fig. 5C).
Discussion In the present study, we have cloned and characterized a novel Rab7 homologue, Rab7b, which is selectively expressed in promyeloid or monocytic leukemia cell lines, and specifically expressed in normal monocytes and monocyte-derived immature DC in peripheral blood. Therefore, Rab7b is expressed in a rather specific pattern and can potentially serve as a new monocytic cell marker. It is shown that phosphoinositide 3-kinase (PI3K), protein kinase B/Akt, and Rab7 were sequentially activated during macropinosome formation [33]. Since conserved motifs involved in PKC and tyrosine kinase phosphorylation, or GTP-binding and -hydrolysis, are found in Rab7b, we predict here that Rab7b is presumably a Rab GTPase that can specifically bind GTP/ GDP and hydrolyze GTP, and possibly regulates protein transport to or from lysosomes under the regulation of kinases, such as PKC and PI3K. Rab proteins are important regulators of protein transport along the endocytic and exocytic pathways [1– 3]. Rab7 has been implicated in the regulation of protein transport from early to late endosomes and it has been used as a late lysosome marker [4–13]. During endocytosis of fluid phase marker horseradish peroxidase (HRP), Rab7 functions in a sequential manner downstream of Rab5, which is associated with early endosomes and involved in the regulation of early endocytosis [34,35]. As Rab7 has been suggested to be associated with controlling of aggregation and fusion of late endocytic structures/lysosomes, it has been implicated in the degradation of proteins and maintenance of the perinuclear lysosome compartment [36]. High homology has been found between Rab7b and Rab7, and Rab7b is associated with lysosomes or acidic pH compartments. Thus, Rab7b may function similar to Rab7, that is, protein transport to or from lysosomes. However, Rab7b possibly only serves as regulator of protein transport in certain cell types or under special conditions, considering that Rab7b is preferentially expressed in DC and CD14þ monocytic cells. DCs and monocytes are potent antigen-presenting cells that can uptake and process exogenous antigens, and present antigens to T cells [37,38]. It is shown that Rab7 plays a role in MHC II molecule-associated antigen presentation [16]. Thus, it is possible that Rab7b is possibly involved in endocytosis and presentation of protein antigens. The roles of Rab7b in antigen uptake and presentation by antigenpresenting cells, such as DC and monocytes/macrophages, are now undergoing.
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Acute promyelocytic leukemia HL-60 and NB4 cells have been used to study leukemia differentiation. After stimulation with PMA, they differentiate mainly towards monocytic lineage, concomitant with adhesion to support matrix, increased phagocytosis capacity, restored respiratory burst, impaired growth rate, and inhibited tumorigenicity [27–32]. Rab7b expression is gradually increased in HL-60 and NB4 cells upon PMA treatments, together with alterations in monocytic differentiation markers, such as upregulation of CD11b and CD14, and downregulation of CD33. It has been demonstrated previously that several Rab proteins, including Rab7, Rab2, and Rab11b, are transcriptionally increased upon PMA treatments [39]. But it remains obscure whether the upregulation of Rab proteins expression is the causing effects of PMA treatments, or the Rab proteins are the effectors that work together to facilitate the restore of the normal functions of leukemia cells. In case of Rab7b upregulation in myeloid leukemia cells after PMA stimulation, a reasonable explanation is that PMA increases Rab7b expression, which in turn promotes the intermediate or late phase differentiation of leukemia cells and restores the function of myeloid cells, such as restored phagocytosis, respiratory burst, and antigen presentation capacity. The mechanisms and the functions of Rab7b in leukemia cell differentiation need further study. However, our studies at least suggest that Rab7b, a novel lysosomal Rab7-like protein, is associated with monocytic differentiation of APL cells.
Acknowledgments This work was supported by grants from the National Key Basic Research Program of China (2001CB510002), the National Natural Science Foundation of China (30121002), and the National High Biotechnology Development Program of China (2002BA711A01). We sincerely thank Qiangsu Guo, Mei Jin, Yan Li, Xianwei Ma, and Xiaoting Zuo for their excellent technical assistance.
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