Regulation of progranulin expression in myeloid cells
Colin H. P. Ong, Zhiheng He, Leonid Kriazhev,
Xiaochuan Shan, Roger G. E. Palfree, and Andrew Bateman
Endocrine Research Laboratories, Department of Medicine, Royal
Victoria Hospital, McGill University, Montreal, Quebec, Canada
Submitted 24 August 2005; accepted in final form 21 July 2006
Ong, Colin H. P., Zhiheng He, Leonid Kriazhev, Xiaochuan
Shan, Roger G. E. Palfree, and Andrew Bateman. Regulation of
progranulin expression in myeloid cells. Am J Physiol Regul Integr
Comp Physiol 291: R1602–R1612, 2006. First published July 27,
2006; doi:10.1152/ajpregu.00616.2005.—Progranulin (pgrn; granu-
lin-epithelin precursor, PC-cell-derived growth factor, or acrogranin)
is a multifunctional secreted glycoprotein implicated in tumorigenesis,
development, inflammation, and repair. It is highly expressed in
macrophage and monocyte-derived dendritic cells. Here we investi-
gate its regulation in myeloid cells. All-trans retinoic acid (ATRA)
increased pgrn mRNA levels in myelomonocytic cells (CD34?pro-
genitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-
60; macrophage RAW 264.7) but not in nonmyeloid cells tested.
Interleukin-4 impaired basal expression of pgrn in U-937. Differen-
tiation agents DMSO, and, in U-937 only, phorbol ester [phorbol
12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late
in differentiation, suggestive of roles for pgrn in more mature termi-
nally differentiated granulocyte/monocytes rather than during growth
or differentiation. The response of pgrn mRNA to ATRA differs in
U-937 and HL-60 lineages. In U-937, ATRA and chemical differen-
tiation agents greatly increased pgrn mRNA stability, whereas, in
HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregu-
lation of pgrn mRNA after stimulation with ATRA was independent
of de novo protein synthesis in U-937 but not HL-60. Chemical
blockade of nuclear factor-?B (NF-?B) activation impaired ATRA-
stimulated pgrn expression in HL-60 but not U-937, whereas in U-937
it blocked PMA-induced pgrn mRNA expression, suggestive of cell-
specific roles for NF-?B in determining pgrn mRNA levels. We
propose that: 1) ATRA regulates pgrn mRNA levels in myelomono-
cytic cells; 2) ATRA acts in a cell-specific manner involving the
differential control of mRNA stability and differential requirement for
NF-?B signaling; and 3) elevated pgrn mRNA expression is charac-
teristic of more mature cells and does not stimulate differentiation.
granulin; stem cell factor; colony-stimulating factor
THE GRANULIN-EPITHELIN precursor progranulin (pgrn; see Ref.
4), which is also called proepithelin (47), PC-cell-derived
growth factor (66), or acrogranin (1), is a multifunctional
regulatory protein that promotes mitosis, survival, and migra-
tion in many cell types (43). It stimulates growth factor-related
signaling pathways such as the phosphorylation of shc, p44/42
mitogen-activated protein kinase, phosphatidylinositol 3-ki-
nase, protein kinase B/AKT, and the p70S6kinase (24, 35, 65)
and contributes to carcinogenesis in breast (51), ovarian (16,
26), renal (18), hepatocellular (10), and prostate (44) cancers,
gliomas (33), and multiple myelomas (63). Physiologically,
pgrn is involved in wound repair (25, 67) and is expressed
during development (8) where it regulates cavitation in preim-
plantation embryos (17), blastocyst hatching (48), and male-
specific differentiation of the neonatal hypothalamus (56, 58).
The granulin (GRN) gene is highly expressed in monocyte-
derived cells, being the 17th and 30th most abundant transcript
in human macrophage (9) and monocyte-derived dendritic cells
(21), respectively. Peptides derived from pgrn, the granulin/
epithelins (grn/epi), were isolated from neutrophils (2), and
GRN is transiently upregulated in neutrophils exposed to Esch-
erichia coli K12 (55). Microarray studies show that GRN is
expressed in CD34?bone marrow cells (20) and is upregulated
in acute myeloid leukemia blasts vs. normal progenitor CD34?
cells (62). Moreover, in the periphery, it is expressed during
chemokine-stimulated alveolar trafficking of monocytes (52),
in inflammatory arthritis but not osteoarthritis (27), during
injury to the spinal cord and brain (37, 38), and in a zebrafish
model of chronic tuberculosis (39).
Functions for GRN in inflammation and repair have been
reported only recently (25, 67). The pgrn inhibits several
actions of tumor necrosis factor-? on neutrophils, including the
respiratory burst, by inhibiting the cytoplasmic kinase pyk-2
(67). Neutrophil elastase cleaves pgrn into grn/epi peptides that
stimulate the release of interleukin (IL)-8 from epithelial cells
(41, 67). The switch between intact pgrn and the grn/epi peptides
is regulated by secreted leukocyte protease inhibitor (SLPI),
which prevents pgrn degradation to grn/epi peptides (41, 67).
Applying pgrn to wounds increases leukocyte inflitration, but
probably indirectly, since it is not chemotactic for neutrophils
(25). In damaged tissue, the secretion of pgrn by inflammatory
cells may assist in repair by stimulating the proliferation and
migration of fibroblasts and endothelial cells (25).
Given that the pgrn mRNA is very highly expressed by
human monocyte-derived cells (9, 21), is present in the bone
marrow (4), is in myeloid leukemic cells that are undifferen-
tiated precursors of granulocytes and monocytes (4, 15), and is
in mature myeloid cells (9, 25), we hypothesized that the pgrn
transcript may be modulated in myeloid cells by cytokines and
hormones that regulate the function of these cells and their
development. Little is known of the factors that regulate pgrn
mRNA expression in myelomonocytic development. Here, we
identify positive and negative regulators of GRN expression
and study its regulation during myeloid cell differentiation.
Address for reprint requests and other correspondence: A. Bateman, Rm.
L2.05, Endocrine Labs, Royal Victoria Hospital, 687 Pine Ave. West, Mon-
treal, Quebec, Cananda H3A 1A1 (e-mail: email@example.com).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Regul Integr Comp Physiol 291: R1602–R1612, 2006.
First published July 27, 2006; doi:10.1152/ajpregu.00616.2005.
0363-6119/06 $8.00 Copyright © 2006 the American Physiological Society http://www.ajpregu.orgR1602
MATERIALS AND METHODS
Materials. Phorbol 12-myristate,13-acetate (PMA), protein kinase
C (PKC) inhibitor, RO-31–8425, genistein, pertussis toxin, and nu-
clear factor-?B (NF-?B) activation inhibitor 6-amino-4-(4-phenoxy-
phenylethylamino)quinazoline (6-APQ) were obtained from Calbio-
chem (San Diego, CA). DMSO was supplied by ICN Biomedicals
(Costa Mesa, CA). All-trans retinoic acid (ATRA), cycloheximide
(CHX), and actinomycin D were purchased from Sigma (St. Louis,
MO). Cytokines were obtained from Genetics Institute (Cambridge,
MA) except interferon (IFN)-? (Schering Canada, Pointe Claire,
Quebec, Canada), IL-4 (Biosource International, Camarillo, CA), and
granulocyte colony-stimulating factor (G-CSF) and granulocyte-mac-
rophage colony-stimulating factor (GM-CSF; Leinco Technologies,
St. Louis, MO). Recombinant human erythropoietin (EPO) was a gift
from Dr. F. Congote (Department of Medicine, Royal Victoria Hos-
pital, Montreal, Quebec, Canada).
Cell culture. Cell lines were purchased from the American Type
Culture Collection (ATCC, Manassas, VA) and were maintained in
the supplier’s recommended medium containing 10% FBS (Biomedia
Canada, Drummondville, Quebec, Canada). The human bone marrow
CD34?hematopoietic progenitor cells were obtained from Cambrex
Bio Science (Walkersville, MA). Human dermal fibroblasts, TF-1,
and HL-60R cells were gifts from Dr. A. Philip (Department of
Surgery, Montreal General Hospital, Montreal, Quebec, Canada) and
Dr. F. Congote and Dr. S. Collins (Fred Hutchinson Cancer Research
Center, Seattle, WA), respectively.
Cell lines of hematopoietic origin were seeded at a density of 106
cells/ml. In the initial screen of pgrn mRNA regulation in U-937 cells,
concentrations of the cytokines used were as follows: IFN-? (1,000
U/ml), IL-1? (10 U/ml), IL-2 (2.5 U/ml), IL-11 (1,000 ng/ml), stem
cell factor (SCF; 10 U/ml), macrophage colony-stimulating factor
(100 ng/ml), GM-CSF (50 ng/ml), IL-3 (100 U/ml), IL-6 (100 U/ml),
G-CSF (1,000 U/ml), IFN-? (100 U/ml), IL-4 (100 U/ml), and IL-5
(50 ng/ml). EPO was used at a final concentration of 5 U/ml. ATRA
was used at 1 ?M. CHX was used at 10 ?g/ml. DMSO was used at
a concentration of 1.25% (vol/vol). PMA was used at 100 nM. For
chemical inhibitors, cells were pretreated with either RO-31–8425,
6-APQ, or CHX for 30 min before the addition of any differentiation
stimulus. Actinomycin D was used at a final concentration of 2 ?g/ml.
Human hematopoietic colony-forming cell assay. Human bone
marrow CD34?hematopoietic progenitor cells were seeded in meth-
ylcellulose-based media (MethoCult, StemCell Technologies, Van-
couver, British Columbia, Canada) containing h (human) SCF, hGM-
CSF, hIL-3, hIL-6, hG-CSF, and hEPO at a density of 700 cells/ml.
ATRA was added to the suspension to final concentrations of either
10?7or 10?6M. The suspensions were then plated in 35-mm dishes
and maintained at 37°C, 5% CO2 for 14 days. Colonies were then
observed through an inverted microscope and enumerated.
Real-time RT-PCR assay on human bone marrow CD34?hema-
topoietic progenitor cells. Individual colony-forming unit granulo-
cyte-macrophage (CFU-GM) colonies were picked and pooled (?30
colonies) for total RNA extraction using Tri-Zol Reagent (GIBCO-
BRL/Life Technologies). The RNA was treated with RQ1 DNase
(Promega, Madison, WI) and reverse transcribed using moloney
murine leukemia virus RT (GIBCO-BRL/Life Technologies). For
quantitative real-time PCR, the primer pairs used for the pgrn tran-
script were 5?-GGACAGTACTGAAGACTCTG-3? (forward primer)
and 5?-GGATGGCAGCTTGTAATGTG-3? (reverse primer), whereas
the primers for ?-actin were 5?-GAAGTGTGACGTGGACATCC-3?
(forward primer) and 5?-CCGATCCACACGGAGTACTT-3? (reverse
primer; GIBCO-BRL/Life Technologies). Amplification reaction mix-
tures were prepared according to the manufacturer’s instructions using a
LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied
Science, Penzberg, Germany) with a final primer concentration of 0.5
?M for each reaction. The amplifications, in duplicates, were performed
in the LightCycler (Roche Applied Science) using the following condi-
tions: hot start step (denaturation) at 95°C for 10 min followed by 45
cycles of 95°C for 10 s, 66°C for 10 s, and 72°C for 10 s. The expression
of GRN was normalized to that of ?-actin. Gene expression was ex-
pressed as N-fold differences where: N ? 2?Cpfor GRN ? ?Cpfor ?-actin; ?Cpis
the difference between the crossing point values for ATRA-treated
and untreated CD34? hematopoietic progenitor cells.
Isolation of total RNA and Northern blotting. Total RNA was
isolated from 107cells using Tri-Zol Reagent. RNA samples, 10 or 15
?g, were denatured with glyoxal at 50°C for 1 h and subjected to
electrophoresis using a 1% agarose gel in 10 mM NaH2PO4, pH 7.
Northern blot analysis was then performed as described previously
(4). Densitometry was performed using Scion Image software and
normalized to the 28S ribosomal RNA bands. A relative density of
one was assigned to the control sample. Values were presented as
mean values ? SE. Statistical analyses were performed using the
Student-Newman-Keuls test (Graphpad Instat software).
Isolation of recombinant human pgrn. Recombinant human pgrn
was made by transient transfection of pgrn/pcDNA3 in COS-7 cells and
purified using a C4 reversed-phase HPLC column as described previ-
ously (23). The biological activity of pgrn was monitored by its ability to
promote the growth of SW-13 cells as described previously (23).
Cell differentiation and cytofluorimetric analysis. Cellular differ-
entiation status was assessed by morphologically scoring 100 cells at
each time point using the Wright-Giemsa staining method (34). The
induction of CD11b was determined using 106cells resuspended in a
total volume of 50 ?l with PBS/0.1% NaN3/1% FBS. After being
blocked with normal human AB serum (Sigma) and normal mouse
IgG (Caltag Laboratories, Burlingame, CA), the cell suspension was
incubated with 20 ?l of phycoerythrin-conjugated anti-CD11b (BD
Biosciences, Mississauga, Ontario, Canada) at 4°C for 45 min in the
dark. The cells were then washed two times and resuspended in 1%
paraformaldehyde in PBS/0.1% NaN3 and analyzed for CD11b by
cytofluorimetry (Clinical Research Institute of Montreal).
Western blot analysis for pgrn. Cells (106cells/ml) in serum- and
phenol red-free RPMI-1640 medium were exposed to either 1 ?M
ATRA, 100 nM PMA, or 1.25% (vol/vol) DMSO for 48 h. Condi-
tioned medium (CM) and cell pellets were collected by centrifugation
at 170 g at room temperature for 5 min. Samples were extracted with
an equal volume of 2? (CM) or 10 ml 1? (cell pellet) extraction
medium [1X: 1 M HCl, 0.17 M NaCl, 1% (vol/vol) trifluoroacetic
acid, and 4.5% (vol/vol) formic acid]. The CM extraction was mixed
well, and the cell pellet was subjected to sonication using an ultrasonic
homogenizer (Cole Palmer Instruments, Chicago, IL). The mixtures
were then centrifuged at 2,000 g at 4°C for 25 min to remove debris.
Extracts were desalted and concentrated using a Waters C18Sep-Pak
cartridge (Milford, MA) as described previously (3) and lyophilized.
(The efficient adsorption of pgrn to C18 cartridges was confirmed
using recombinant human pgrn.) The samples were resuspended in
milliQ water and a volume equivalent to 3.0 ? 106cells was subjected
to SDS-PAGE (12%). After protein transfer to nitrocellulose mem-
branes (Hybond-C extra; Amersham Pharmacia Biotech, Baie d’Urfe,
Quebec, Canada), the membranes were blocked with 5% nonfat milk
and probed with a rabbit polyclonal anti-human pgrn antibody (1:
10,000; prepared by Dr. G. Sadvakassova, Royal Victoria Hospital)
followed by donkey anti-rabbit antibody conjugated to horseradish
peroxidase (1:5,000). Antigen was visualized by chemiluminescence
using luminol as substrate (Amersham Pharmacia Biotech). Identical
duplicate blots were probed with preimmune serum.
Regulation of pgrn mRNA levels by retinoic acid. The
monoblastoid U-937 cell line can be stimulated to differentiate
toward more mature monocyte-like cells (22, 42) or adherent
macrophage-like cells (40). When we exposed U-937 cells to a
panel of cytokines and other physiological mediators that
regulate myeloid maturation or function, ATRA stimulated a
REGULATION OF PROGRANULIN EXPRESSION IN MYELOID CELLS
AJP-Regul Integr Comp Physiol • VOL 291 • DECEMBER 2006 • www.ajpregu.org
44. Pan CX, Kinch MS, Kiener PA, Langermann S, Serrero G, Sun L,
Corvera J, Sweeney CJ, Li L, Zhang S, Baldridge LA, Jones TD, Koch
MO, Ulbright TM, Eble JN, and Cheng L. PC cell-derived growth
factor expression in prostatic intraepithelial neoplasia and prostatic ade-
nocarcinoma. Clin Cancer Res 10: 1333–1337, 2004.
45. Paya CV, Ten RM, Bessia C, Alcami J, Hay RT, and Virelizier JL.
NF-kappa B-dependent induction of the NF-kappa B p50 subunit gene
promoter underlies self-perpetuation of human immunodeficiency virus
transcription in monocytic cells. Proc Natl Acad Sci USA 89: 7826–7830,
46. Pesole G, Mignone F, Gissi C, Grillo G, Licciulli F, and Liuni S.
Structural and functional features of eukaryotic mRNA untranslated re-
gions. Gene 276: 73–81, 2001.
47. Plowman GD, Green JM, Neubauer MG, Buckley SD, McDonald VL,
Todaro GJ, and Shoyab M. The epithelin precursor encodes two proteins
with opposing activities on epithelial cell growth. J Biol Chem 267:
48. Qin J, Diaz-Cueto L, Schwarze JE, Takahashi Y, Imai M, Isuzugawa
K, Yamamoto S, Chang KT, Gerton GL, and Imakawa K. Effects of
progranulin on blastocyst hatching and subsequent adhesion and out-
growth in the mouse. Biol Reprod 73: 434-442, 2005.
49. Robertson KA, Emami B, and Collins SJ. Retinoic acid-resistant HL-
60R cells harbor a point mutation in the retinoic acid receptor ligand-
binding domain that confers dominant negative activity. Blood 80: 1885–
50. Rovera G, Santoli D, and Damsky C. Human promyelocytic leukemia
cells in culture differentiate into macrophage-like cells when treated with
a phorbol diester. Proc Natl Acad Sci USA 76: 2779–2783, 1979.
51. Serrero G and Ioffe OB. Expression of PC-cell-derived growth factor in
benign and malignant human breast epithelium. Hum Pathol 34: 1148–
52. Srivastava M, Jung S, Wilhelm J, Fink L, Buhling F, Welte T, Bohle
RM, Seeger W, Lohmeyer J, and Maus UA. The inflammatory versus
constitutive trafficking of mononuclear phagocytes into the alveolar space
of mice is associated with drastic changes in their gene expression profiles.
J Immunol 175: 1884–1893, 2005.
53. Steffan NM, Bren GD, Frantz B, Tocci MJ, O’Neill EA, and Paya CV.
Regulation of IkB alpha phosphorylation by PKC- and Ca(2?)-dependent
signal transduction pathways. J Immunol 155: 4685–4691, 1995.
54. Stoeckle MY. Post-transcriptional regulation of gro alpha, beta, gamma,
and IL-8 mRNAs by IL-1 beta. Nucleic Acids Res 19: 917–920, 1991.
55. Subrahmanyam YV, Yamaga S, Prashar Y, Lee HH, Hoe NP, Kluger
Y, Gerstein M, Goguen JD, Newburger PE, and Weissman SM. RNA
expression patterns change dramatically in human neutrophils exposed to
bacteria. Blood 97: 2457–2468, 2001.
56. Suzuki M, Bannai M, Matsumuro M, Furuhata Y, Ikemura R,
Kuranaga E, Kaneda Y, Nishihara M, and Takahashi M. Suppression
of copulatory behavior by intracerebroventricular infusion of antisense
oligodeoxynucleotide of granulin in neonatal male rats. Physiol Behav 68:
57. Suzuki M, Yonezawa T, Fujioka H, Matuamuro M, and Nishihara M.
Induction of granulin precursor gene expression by estrogen treatment in
neonatal rat hypothalamus. Neurosci Lett 297: 199–202, 2001.
58. Suzuki M, Yoshida S, Nishihara M, and Takahashi M. Identification of
a sex steroid-inducible gene in the neonatal rat hypothalamus. Neurosci
Lett 242: 127–130, 1998.
59. Suzuki Y, Shimada J, Shudo K, Matsumura M, Crippa MP, and
Kojima S. Physical interaction between retinoic acid receptor and Sp1:
mechanism for induction of urokinase by retinoic acid. Blood 93: 4264–
60. Tobe M, Isobe Y, Tomizawa H, Nagasaki T, Takahashi H, and
Hayashi H. A novel structural class of potent inhibitors of NF-kappa B
activation: structure-activity relationships and biological effects of 6-ami-
noquinazoline derivatives. Bioorg Med Chem 11: 3869–3878, 2003.
61. Vassiliadis S, Kyrpides N, and Papamatheakis J. The role of IL-4 in
human myeloid leukemia: stimulation of RNA synthesis and transduction
of differentiation signals through an IL-4 receptor leads to functional and
HLA positive HL-60 cells. Leuk Lymphoma 7: 235–242, 1992.
62. Virtaneva K, Wright FA, Tanner SM, Yuan B, Lemon WJ, Caligiuri
MA, Bloomfield CD, de La Chapelle A, and Krahe R. Expression
profiling reveals fundamental biological differences in acute myeloid
leukemia with isolated trisomy 8 and normal cytogenetics. Proc Natl Acad
Sci USA 98: 1124–1129, 2001.
63. Wang W, Hayashi J, Kim WE, and Serrero G. PC cell-derived growth
factor (granulin precursor) expression and action in human multiple
myeloma. Clin Cancer Res 9: 2221–2228, 2003.
64. Witcher M, Ross DT, Rousseau C, Deluca L, and Miller WH Jr.
Synergy between all-trans retinoic acid and tumor necrosis factor path-
ways in acute leukemia cells. Blood 102: 237–245, 2003.
65. Zanocco-Marani T, Bateman A, Romano G, Valentinis B, He ZH, and
Baserga R. Biological activities and signaling pathways of the granulin/
epithelin precursor. Cancer Res 59: 5331–5340, 1999.
66. Zhou J, Gao G, Crabb JW, and Serrero G. Purification of an autocrine
growth factor homologous with mouse epithelin precursor from a highly
tumorigenic cell line. J Biol Chem 268: 10863–10869, 1993.
67. Zhu J, Nathan C, Jin W, Sim D, Ashcroft GS, Wahl SM, Lacomis L,
Erdjument-Bromage H, Tempst P, Wright CD, and Ding A. Conver-
sion of proepithelin to epithelins: roles of SLPI and elastase in host
defense and wound repair. Cell 111: 867–878, 2002.
REGULATION OF PROGRANULIN EXPRESSION IN MYELOID CELLS
AJP-Regul Integr Comp Physiol • VOL 291 • DECEMBER 2006 • www.ajpregu.org