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Monash Institute of Reproduction and Development, Monash University, Clayton,Australia- R" W* ~2 L' F6 L# i% M5 t) n
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Key Words. Human embryonic stem cells ? Gap junction ? Connexin 43 ? Connexin 45
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3 n. L& U+ W/ A6 x3 Z" QCorrespondence: Dr. A. P谷bay, Ph.D., Monash Institute of Reproduction and Development, Monash University, 246 Clayton Road, Clayton, VIC 3168,Australia. Telephone: 0061-395947302; Fax: 0061-95947311, e-mail: alice.pebay@med.monash.edu.au* R# l1 O0 m# \ j3 e
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9 K6 P/ P3 {) i% e# U- Z9 K0 c+ \! U8 iGap junctions are intercellular channels that consist of two hemi-channels, termed connexons, each localized in the membrane of adjacent cells. Each connexon consists of six integral membrane proteins, termed connexins . Gap junction intercellular communication (GJIC) allows cell–cell exchange of inorganic salts and small metabolites of less than ~1 kDa with a maximum diameter of ~1.5 nm . Such intercellular coupling has been implicated in the control of various cellular processes, including intercellular buffering of cytoplasmic ions, electrical synchronization, control of cell migration, cell proliferation, cell differentiation, metabolism, and apoptosis .4 N" j2 N- S4 L
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Gap junctions have long been implicated in cellular growth control. Although the molecular mechanisms remain largely unknown, it is generally believed that upregulation of GJIC is associated with inhibition of cellular growth, whereas downregulation of GJIC correlates with stimulation of growth . Most, if not all, cancer cells lack functional GJIC . A recent hypothesis suggests that GJIC deficiency is also a characteristic of stem cells . This hypothesis is based on the fact that two types of epithelial adult stem cell, keratinocyte stem cells and corneal epithelial stem cells , do not express connexin and are GJIC deficient. Studies on several presumptive adult progenitor cells (including immortalized and transformed cells) also support this hypothesis, because these different cell types also lack functional gap junctions . However, gap junctions have not been studied extensively in embryonic stem (ES) cells. It has been reported that connexin 43 and connexin 45 mRNAs are expressed in undifferentiated mouse ES cells .2 @. U$ h B7 h% X% W
- I0 M( t+ i7 ?, C; t* phESCs are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts . ES cells can be grown in vitro indefinitely while maintaining a normal karyotype, and throughout long periods of cultivation in vitro they remain pluripotent, processing the ability to develop into multiple cell types representative of all three embryonic germ layers and extra-embryonic tissues . This work aimed to study the expression of connexins in hESCs and to investigate the presence of functional gap junctions within hESC colonies.4 z ~4 r# q5 z* f# P3 O
. H. C8 d& d) QMATERIALS AND METHODS
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& G( U* d2 O8 F$ M' p. ?5 s) B0 G) TBoth HES-3 and HES-4 cells expressed mRNA transcripts for connexin 43 and connexin 45 (Fig. 1A). Expression of the connexin proteins in stem cells was confirmed by costaining cultures with the stem cell surface antigen GCTM-2 and antibodies against the two connexins (Figs. 2, 3). Although con-nexin 43 was mainly localized at the cell surface (Fig. 2), connexin 45 was mostly cytoplasmic (Fig. 3). Because the antibodies used are capable of recognizing the mouse con-nexins 43 and 45, we can conclude that MEF cells do not express detectable levels of these proteins under these conditions (Figs. 2, 3). Because there are three phosphorylated forms of connexin 43 in other cell types, we checked its phosphorylated state in hESCs. Immunoblot analysis with an antibody reactive with all of the phosphorylated forms of connexin 43 revealed the presence of a triplet of bands representing the nonphosphorylated (NP) and the phosphorylated (P1, P2) forms of connexin 43 (Fig. 1B). These results also indicate that connexin 43 is mostly NP in hESCs (Fig. 1B).
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Figure 1. (A): Messenger RNA expression of connexin 43 and connexin 45 in HES-3 and HES-4 cells. Control reactions omitting the addition of reverse transcriptase or primers (-ve control) were all negative. Sequencing of polymerase chain reaction products confirmed identity of human transcript without mutation. (B): Western blot analysis of connexin 43 in HES-3 cells. Abbreviations: Cx, connexin; NP, nonphosphorylated; P1 and P2, phosphorylated.
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Figure 2. Expression of connexin 43 in human embryonic stem cells. Immunostaining of HES-3 cells (A) with Hoechst 33342 (B), connexin 43 (C), and GCTM2 (D). Higher magnification of HES cells stained with connexin 43 (E). Merging image of HES-3 cells and MEF dually immunostained with connexin 43 and GCTM2 (F). Scale bars = 50 μm. Abbreviation: MEF, mouse embryonic fibroblast.. Z* ?( P# ^; f
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Figure 3. Expression of connexin 45 in human embryonic stem cells. Immunostaining of HES-3 cells (A) with Hoechst 33342 (B), connexin 45 (C), and GCTM2 (D). Merging image of HES-3 cells and MEF dually immunostained with connexin 45 and GCTM2 (E). Scale bars = 50 μm. Abbreviation: MEF, mouse embryonic fibroblast., `3 F4 [+ M x9 h
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We next examined the presence or absence of functional gap junctions in hESCs using the scrape loading/dye transfer assay with Lucifer yellow and rhodamine-dextran. Because of its low molecular weight (522 Da), Lucifer yellow diffuses from cell to cell through functional gap junctions. On the other hand, rhodamine-dextran (10,000 Da) is too large to diffuse through gap junctions and thus serves as a negative control to confirm that the Lucifer yellow transfer is solely due to gap junction coupling and not due to membrane fusions or formation of cytoplasmic bridges. When hESCs were scraped and incubated in presence of these fluorescent dyes, we observed extensive Lucifer yellow diffusion through hESC colonies, whereas rhodamine-dextran remained at the site of the scrape injury (Figs. 4A–C). Control colonies incubated with either marker in the absence of scraping demonstrated no uptake or dye transfer of Lucifer yellow or rhodamine-dextran (data not shown). Moreover, we did not observe any Lucifer yellow diffusion between hESCs and the supportive feeder cells (Fig. 4). The Lucifer yellow diffusion in hESCs was not affected by the absence of Ca2 Mg2 (Figs. 4D–F) but was inhibited by PMA, a protein kinase C (PKC) activator, and by the extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 (Figs. 4G–L). Altogether, these data demonstrate that hESCs are coupled through functional gap junctions and suggest that these gap junctions are not regulated by Ca2 Mg2 . These results also suggest that activation of PKC, as well as inhibition of ERK phosphorylation, inhibit GJIC in hESCs.! N, x2 N4 Y1 E% C( D( ]6 M
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Figure 4. Gap junctional intercellular communication in human embryonic stem cells. (A, D, G, J): Light and fluorescence micrographs with Lucifer yellow (B, E, H, K) and rhodamine-dextran (C, F, I, L) in HES-3 cells. Rhodamine-dextran was used as a negative control, showing no dye transfer across to the neighboring cell. Cells were incubated in the presence (A–C) or absence (D–F) of Ca2 Mg2 or in the presence of phorbol 12-myristate 13-acetate (G–I) or U0126 (J–L). Scale bars = 100 μm.
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DISCUSSION
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This study was supported by Monash University, ES Cell International, and the National Institutes of Health (NIGMS GM68417).
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Raymond Wong and Alice P谷bay contributed equally to this work
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REFERENCES
) p) o& C$ N7 O* Q- W, f: N) G/ ^$ z* l* K, ~; d
Willecke K, Hennemann H, Dahl E et al. The diversity of connexin genes encoding gap junctional proteins. Eur J Cell Biol 1991; 56:1–7.
* a. f0 @! l8 S
) a5 }0 ?/ [2 B( r+ lBruzzone R, White TW, Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem 1996;238:1–27.! W+ y/ m; C# R4 P0 g
! F, M |7 r1 V
Simon AM, Goodenough DA. Diverse functions of vertebrate gap junctions. Trends Cell Biol 1998;8:477–483.6 Z1 D! O( L9 V& ]
% i1 a% v; W5 a& N& c# b7 C% v
De Maio A, Vega V, Contreras J. Gap junctions, homeostasis, and injury. J Cell Physiol 2002;191:269–282.' m+ N- `) J- l$ W( n3 v9 P( o! D. Q
& @" `& ~, R- N; s
Loewenstein WR, Rose B. The cell-cell channel in the control of growth. Semin Cell Biol 1992;3:59–79.6 X8 l7 D8 W) r# p1 j- I! z
( G7 H! D @. F6 p# S# mTrosko JE, Chang CC. Mechanism of up-regulated gap junctional intercellular communication during chemoprevention and chemotherapy of cancer. Mutat Res 2001;480–481:219–229.& q# v* q& J1 |+ W+ q
7 N/ T5 A& B# x+ e7 q3 J; r3 W3 O+ B
Trosko JE, Chang CC. Isolation and characterization of normal adult human epithelial pluripotent stem cells. Oncol Res 2003;13:353–357.
2 C# Q' W; ^: n1 X3 A) O1 B' a, k* |6 ~" w4 }4 |# c' j6 R
Trosko JE, Chang CC, Wilson MR et al. Gap junctions and the regulation of cellular functions of stem cells during development and differentiation. Methods 2000;20:245–264.
1 E) `1 F H' b9 O8 ^
' y F# ]( m0 ^Trosko JE. Human stem cells as targets for the aging and diseases of aging processes. Med Hypotheses 2003;60:439–447.
; n5 i+ h. T% c+ ? ]9 B5 }* S$ `/ ?- [
Matic M, Evans WH, Brink PR et al. Epidermal stem cells do not communicate through gap junctions. J Invest Dermatol 2002;118:110–116.
! l5 l9 s: _/ `$ R/ `" Y- H. Y; N5 c g" C
Matic M, Petrov IN, Chen S et al. Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity. Differentiation 1997;61:251–260.+ w' Q1 I- w% J" P6 ?! K
/ G9 }7 |7 f q. I8 t8 u
Tai MH, Olson LK, Madhukar BV et al. Characterization of gap junctional intercellular communication in immortalized human pancreatic ductal epithelial cells with stem cell characteristics. Pancreas 2003;26:e18–e26., V. h& b8 ]) @# J) P8 c- X
6 n9 u. p; {" f" ?: k4 o2 n) x5 L
Dowling-Warriner CV, Trosko JE. Induction of gap junctional intercellular communication, connexin43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway. Neuro-science 2000;95:859–868.
, ^( S) {5 \! Z1 o# b0 F0 T- s* s+ H
Holland MS, Tai MH, Trosko JE et al. Isolation and differentiation of bovine mammary gland progenitor cell populations. Am J Vet Res 2003;64:396–403.7 i4 a6 r$ v7 o `) s2 Q0 l
1 X" ]! S6 U) c9 ?; W* gKao CY, Nomata K, Oakley CS et al. Two types of normal human breast epithelial cells derived from reduction mammoplasty: phenotypic characterization and response to SV40 transfection. Carcinogenesis 1995;16:531–538.: H& F9 \/ z" X G. J1 F; R1 {; V
! L2 O# k, [- D$ J* z0 ? K8 R# @* [Chang CC, Trosko JE, el-Fouly MH et al. Contact insensitivity of a subpopulation of normal human fetal kidney epithelial cells and of human carcinoma cell lines. Cancer Res 1987;47:1634–1645.% E) Y6 F7 U* L6 ?) Z y2 a
9 u) u; m- [0 {Oyamada Y, Komatsu K, Kimura H et al. Differential regulation of gap junction protein (connexin) genes during car-diomyocytic differentiation of mouse embryonic stem cells in vitro. Exp Cell Res 1996;229:318–326.
, Q* C% C7 S/ _. X6 Y/ t( L6 n" p% c% Q2 j
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
0 l9 Y' L7 H# V) u% A6 B- l; v4 i
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.9 {. {: c/ n8 T. l3 V
2 C8 z" w! p, @( x. K2 Y) N) PPera MF, Reubinoff B, Trounson A. Human embryonic stem cells. J Cell Sci 2000;113(pt 1):5–10.
4 n2 t4 A' J) F. J2 R$ m9 p' ^/ R" h* ]" f& o4 e& m8 F
Pera MF, Filipczyk AA, Hawes SM et al. Isolation, characterization, and differentiation of human embryonic stem cells. Methods Enzymol 2003;365:429–446.
" i: _! ^' q8 X4 I# ?, ^( l! C- `& P1 s3 L& \ y) X
Wolvetang EJ, Wilson TJ, Sanij E et al. ETS2 overexpression in transgenic models and in Down syndrome predisposes to apoptosis via the p53 pathway. Hum Mol Genet 2003;12:247–255.
" _3 Y1 p+ D+ h; \# P- J9 ?3 U% m F# W: E( N
Nishi M, Kumar NM, Gilula NB. Developmental regulation of gap junction gene expression during mouse embryonic development. Dev Biol 1991;146:117–130.
# }1 i3 a' P( f4 K9 v5 \
& H/ @! f8 }7 T: W0 X/ ?Belliveau DJ, Bechberger JF, Rogers KA et al. Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells. Dev Genet 1997;21:187–200.
5 }. y/ `" `! m* A7 d0 G! O/ y( L' Z9 D8 ]" O
Bani-Yaghoub M, Bechberger JF, Naus CC. Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells. J Neurosci Res 1997;49:19–31.
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Hennemann H, Schwarz HJ, Willecke K, Characterization of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs. Eur J Cell Biol 1992;57:51–58.8 A2 ^% }6 F$ }) k8 ]
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Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.
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; y9 C1 f N( s# S$ g2 @, J+ q) [2 VLampe PD, Lau AF. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys 2000; 384:205–215.(Raymond C.B. Wong, Alice ) |
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发表于 2009-3-5 10:36
发表于 2015-6-5 20:49
