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Regular Articles |
From the Walter and Eliza Hall Institute*
and the
Rotary Bone Marrow Research Laboratories,
Royal Melbourne Hospital, Melbourne, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis is an evolutionarily conserved process that is characterized morphologically by cell shrinkage, plasma membrane blebbing, and chromatin condensation.7 Cell death can be induced by a variety of physiological as well as experimentally applied stimuli that activate distinct signaling pathways. These pathways ultimately converge on a common effector machinery that is driven by a family of cysteine proteases (caspases) that cleave substrates after aspartate residues.8,9 Caspases exist in living cells as zymogens with low enzymatic activity. They need to be cleaved at aspartate residues to generate fragments of ~20 kd and ~10 kd that are assembled into the fully active tetrameric (p202p102) enzyme.9 Adaptor proteins, such as mammalian Apaf-1 or FADD (also called MORT1) and Caenorhabditis elegans CED-4, promote aggregation and self-processing of so-called initiator caspases.10,11 These in turn proteolytically activate so-called effector caspases, giving rise to the proteolytic avalanche that culminates in the degradation of vital cellular constituents and cell collapse.
The Bcl-2 protein family regulates pathways to apoptosis that are activated by growth factor deprivation or many forms of intracellular damage but play little role in apoptosis induced by tumor necrosis factor receptor family members, at least in lymphocytes.12,13 Pro-survival Bcl-2 proteins, including mammalian Bcl-2, Bcl-xL, Bcl-w, A1/Bfl1, Mcl-1, Boo/Diva, as well as C. elegans CED-9, inhibit apoptosis by blocking the activity of adaptor proteins, such as mammalian Apaf-1 or C. elegans CED-4.12 The proapoptotic members of the Bcl-2 family can antagonize Bcl-2 and its homologs. Based on their structure, these proapoptotic proteins can be further subdivided into two groups. One includes mammalian Bax, Bcl-xS, Bak, and Bok/Mtd, which share two or three regions of homology (BH regions) with Bcl-2, whereas the other subgroup, including mammalian Bad, Bik/Nbk, Bid, Harakiri/DP5, Blk, and Bim/Bod,12 as well as C. elegans EGL-1,14 only have similarities in the short BH3 domain. The proapoptotic Bcl-2 family members bind via their BH3 domain to Bcl-2 or its functional homologs, and it is believed that this initiates apoptosis by unleashing Apaf-1/CED-4-like adaptors that can then activate certain procaspases (eg, mammalian procaspase-9 or C. elegans CED-3).12
Bim was originally cloned as a Bcl-2-interacting protein by screening a
phage expression library constructed from a mouse thymic
lymphoma.15
Alternative splicing generates three Bim
isoforms, BimS, BimL, and
BimEL, which can all neutralize the activity of
pro-survival Bcl-2-like proteins through their BH3 domain. However, the
three isoforms vary considerably in their pro-apoptotic activity. This
is partly due to sequestration of the less potent forms,
BimL and BimEL, but not
BimS, to cytoskeletal structures via association
with dynein light chain LC8.16
Gene targeting experiments in mice have revealed the essential roles of Bim.17 Most bim-/- embryos died before E9.5, demonstrating that Bim plays a critical role in development. The remaining bim-/- mice were live-born and had an outwardly normal appearance. They had, however, two- to fivefold more lymphocytes, macrophages, and granulocytes, probably because of their increased resistance to cytokine withdrawal-induced apoptosis. This indicates that Bim is required for hematopoietic cell homeostasis. Cell survival experiments with lymphocytes showed that Bim is essential for death after some but not all apoptotic stimuli that can be blocked by Bcl-2. By 1 year of age most live-born bim-/- mice accumulated huge excesses (30–200-fold) of immunoglobulin (Ig)-secreting plasma cells and succumbed to fatal autoimmune glomerulonephritis, demonstrating that Bim imposes an important barrier against autoimmunity.
Initial studies demonstrated that bim mRNA is expressed at low levels in several transformed B- and T-lymphoid cell lines. To further investigate the physiological roles of Bim, we have generated a panel of monoclonal antibodies (mAbs) that specifically recognize distinct Bim isoforms. Here we describe an analysis of the expression of Bim protein and bim mRNA in transformed cell lines and normal mouse tissues. BimL and BimEL were associated with cytoplasmic structures in lymphocytes, myeloid cells, epithelial cells, neuronal cells, and germ cells, but BimS could not be detected in any cell type. These results have implications for cell death control in development and tissue homeostasis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments with animals were performed according to the guidelines of the Royal Melbourne Hospital Research Foundation Animal Ethics Committee. Wistar rats and C57BL/6 mice were obtained from our institute’s breeding facility at Kew (Victoria, Australia). The Bim-deficient knockout (bim-/-) mice have previously been described.17
Expression Constructs and Protein Purification
Expression vectors for EE epitope-tagged mouse
BimS, BimL,
BimEL, BimL
BH3, Bad, and
Bax have previously been described.15,18
The other
expression vectors were constructed by cloning cDNAs for human
BimEL, mouse BimEL (lacking
the internal splice donor and acceptor sites that allow production of
BimL), mouse BimL (aa 2–72
[KO]; gift of P. Bouillet), Bcl-2, Bcl-w, and Bok (gift of Aaron
Hsueh) into pEF PGKpuro or pEF PGKhygro vectors incorporating
the N-terminal epitope tags EE (EYMPME)19
or HA
(YPYDVPDYA).20
Full-length mouse
bimL or bimS
cDNAs were cloned into pGEX (128/128)21
or pQE-9 (Qiagen),
respectively, for the production of BimL (GST
FLAG BimL) or BimS
(His6BimS) proteins in the
bacterial strains BL21 (DE3) pLysS or
SG13009/[pREP4]. The proteins were produced according to the
manufacturers’ protocols (Amersham, Pharmacia, or Qiagen).
Immunization and Hybridoma Fusion
Wistar rats were initially immunized by subcutaneous (s.c.) injection with 100 µg of purified recombinant protein dissolved in complete Freund’s adjuvant (Difco, Detroit, MI). Two subsequent boosts of the immunogen, resuspended in incomplete Freund’s adjuvant (Difco), were injected s.c. 3 and 6 weeks later. A final boost with protein dissolved in phosphate-buffered saline (PBS) was given i.v. and i.p. 4 weeks later. Three days later, hybridomas were generated by fusing spleen cells from immunized rats with the SP2/0 myeloma cell line as previously described.22,23 Hybridomas producing monoclonal antibodies to Bim were identified and their isotype determined by a screening strategy that we have previously described.23 Clones of the interleukin-3 (IL-3)-dependent mouse myeloid cell line FDC-P1 stably expressing Bcl-2 alone, or Bcl-2 plus mouse BimL or BimS, were mixed at a 1:1 ratio, fixed in 1% paraformaldehyde/PBS, permeabilized with 0.3% saponin (Sigma), and stained with hybridoma supernatants. Bound antibodies were revealed with fluorescein isothiocyanate-conjugated goat anti-rat Ig antibodies (Southern Biotechnology) and analyzed in a FACScan analyzer (Becton Dickinson). A single peak of background immunofluorescence indicated that a particular antibody did not recognize Bim, whereas a single peak of high intensity indicated binding to a molecule other than Bim, present in FDC-P1 cells. A double peak histogram, one with background fluorescence intensity and the other with high intensity, identified monoclonal antibodies specific to Bim.23 Hybridomas producing antibodies to Bim were cloned twice and then adapted for growth in low-serum medium. For production of large amounts of antibodies, hybridomas were cultured for several weeks in the miniPERM classic 12.5-kd production and nutrient module (Heraeus). Antibodies were purified on a protein G-Sepharose column (Pharmacia) according to the manufacturer’s protocols.
Cell Lines and Tissue Culture
The cell lines used for analysis of Bim expression are indicated
in Table 1
. Details of those cell lines
not cited in previous studies from this laboratory3,24-29
are available from the authors. These cells were cultured in the
high-glucose version of Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum, 50 µmol/L 2-mercaptoethanol,
13 µmol/L folic acid, and 100 µmol/L L-asparagine or
were grown in Dulbecco’s modified Eagle’s medium or RPMI medium with
10% fetal calf serum only. FDC-P1 and BAF-3 cells or their derivatives
were cultured in the presence of IL-3 (1000 U/ml) produced by X63/0
hybridoma cells stably transfected with an IL-3 expression
construct.30
FDC-P1 clones stably expressing human Bcl-2,
human Bcl-2 plus EE-mouse BimL, or human Bcl-2
plus EE-mouse BimS have been described
previously.15,18
Granulocyte differentiation of
34.6Myl cells was enhanced by the addition of 1.5% dimethyl
sulfoxide.25
Liposome-mediated transfection
(Lipofectamine; Gibco BRL) of 293T cells was performed as previously
described.31
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Cell lines or transfected 293T cells were harvested, washed twice in cold PBS, and lysed in lysis buffer (20 mmol/L Tris/HCl (pH 8.0); 125 mmol/L NaCl; 1 mmol/L EGTA; 1% Triton X-100; 10% glycerol; 0.5 µg/ml Pefabloc; 1 µg/ml of each of leupeptin, aprotinin, soybean trypsin inhibitor, and pepstatin; 5 mmol/L NaF; and 2 mmol/L Na3VO4; all reagents from Sigma or Roche Diagnostics). For the preparation of tissue lysates, organs were excised, washed in PBS, and immediately frozen in isopentane on dry ice and were later homogenized at 4°C in lysis buffer as detailed elsewhere.32
Immunoprecipitation was performed according to previously published protocols.33 Briefly, cell lysates (from 107 cells) or tissue lysates (1.5–3 mg total protein) were precleared by an incubation with a control mAb and protein G-Sepharose before immunoprecipitating with anti-Bim 5E5 or 14A8 mAbs plus protein G-Sepharose for 1.5 hours. After extensive washing (six times in lysis buffer), the immunoprecipitated material was eluted by boiling in sodium dodecyl sulfate-polyacrylamide gel loading buffer, size-fractionated on polyacrylamide gels (Novex), and transferred to nitrocellulose membranes by electroblotting. Nonspecific binding of antibodies to membranes was blocked by incubation overnight in 5% skimmed milk, 1% casein, and 0.05% Tween-20. The membranes were then probed with 5E5 or 14A8 anti-Bim mAbs (1–2 µg/ml), followed by either biotinylated mouse anti-rat IgG2b mAb and horseradish peroxidase (HRP)-conjugated streptavidin (Jackson ImmunoResearch) or HRP-conjugated mouse anti-rat IgG2a mAb (SeroTec). Bound antibodies were visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia). To control for the concentration and integrity of proteins in the tissue lysates, identical blots were probed with mouse anti-HSP70 mAb N6 (a gift from Dr R. Anderson, Peter MacCallum Cancer Research Institute, Melbourne, Australia), followed by HRP-conjugated sheep anti-mouse Ig antibodies (Silenius) and detection by ECL (Amersham Pharmacia).
Immunohistochemistry
Mouse tissues or cell lines were fixed in Histochoice fixative
(Amresco, Solon, OH) and processed for paraffin embedding, cell
lines were resuspended in 1% low-melting-point agarose before
paraffin embedding. Immunohistochemistry was performed according to a
modified protocol detailed elsewhere.34
Paraffin-embedded sections were deparaffinized and gradually
rehydrated. Aldehydes were quenched by incubation in 0.2 mol/L glycine
for 30 minutes, and sections were treated with 0.3% hydrogen peroxide
and 10% methanol to block endogenous peroxidase activity. Nonspecific
staining was prevented by incubation in blocking reagent (TSA indirect
signal amplification; NEN, Boston, MA), and cells were then
permeabilized by incubation in 0.2% Triton X-100 containing 1% bovine
serum albumin. Sections were incubated overnight at 4°C with anti-Bim
mAb 4E4, 5E5, 9F5, or 14A8, or with one of the following
isotype-matched control antibodies: rat IgG2a/
(clone B35–95), rat
IgG2b/ (clone A95–1 or Ter119), or rat IgG1/ (clone R3–34)
(Pharmingen). After they were washed in PBS containing 0.2% Triton
X-100, tissues were incubated with either biotinylated mouse anti-rat
Ig mAb Mar 18.5 or biotinylated mouse anti-rat IgG isotype-specific
antibodies (anti-rat IgG1: RG11/39.4; anti-rat IgG2a: RG7/1.30; or
anti-rat IgG2b: RG7/11.1). After washing, sections were incubated with
HRP-streptavidin (NEN), and the signal was amplified with biotinyl
tyramide according to the manufacturer’s instructions (NEN). This was
followed by a second incubation with HRP-streptavidin. Finally,
sections were stained with diaminobenzidene (Sigma) in 0.07% hydrogen
peroxide, counterstained with hematoxylin, and dehydrated in graded
concentrations of alcohol and histolene before mounting in DPX
(BDH, Poole, UK).
In Situ Hybridization
The full-length mouse bimL cDNA was subcloned into pBSIISK+ (Stratagene). Antisense RNA probes were prepared by in vitro transcription of the pBSIISK+ BimL fragment, using T3 RNA polymerase with digoxigenin-UTP (Roche Diagnostics). Control sense RNA probes were prepared in a similar manner, using T7 RNA polymerase. Standard in situ hybridization was performed as described35 on paraffin-embedded mouse tissue sections that had been fixed in 4% paraformaldehyde.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunization of rats with mouse BimL or
BimS proteins followed by hybridoma fusion
yielded several Bim-specific mAbs.23
Based on our initial
studies we selected two mAbs, 5E5 and 14A8, for further experiments.
Both recognize mouse and human BimL and were
efficacious in many applications, including immunofluorescence
staining,23
Western Blotting (Figure 1, A–F)
, and immunoprecipitation (Figures 2G, 3, and 4)
. Immunoblotting of lysates
from cells transfected with expression constructs encoding different
Bcl-2 family members demonstrated that the two mAbs were highly
specific for Bim. The mAbs bound to mouse and human Bim but did not
detect Bcl-2, Bcl-w, Bcl-xL, Bak, Bax, or the
BH3-only protein Bad (Figure 1, A–C)
. The only cross-reactivity
observed was that mAb 5E5 bound to an unknown 26-kd protein present in
some kidney cell lines (eg, 293T) but not in other cell types (Figure 1A)
.
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, whereas 14A8
recognizes all three isoforms, BimS,
BimL, and BimEL (Figure 1E)
. Importantly, both mAbs bind to an N-terminal fragment (aa 2–72)
and not to the BH3 domain of Bim (Figure 1, D–F)
, the only region in
Bim that is common in related proteins.
We also tested the specificity of mAbs 5E5 and 14A8 in
immunohistochemical staining of cells transfected with expression
constructs encoding BimEL,
BimL, BimS,
BimL lacking the BH3 region
(BimLBH3), Bcl-2, Bad, or Bak, and processed
for paraffin embedding and stained for mAbs 5E5 (Figure 2, A–F)
and
14A8 (not shown). Both mAbs 5E5 and 14A8 showed the same specificity as
seen in Western blotting, immunofluorescent staining, and
immunoprecipitation.
These results show that mAb 14A8 binds to an N-terminal epitope that is
present in all Bim isoforms. In contrast, mAb 5E5 binds to an epitope
that is unique to BimL and is generated by amino
acids spanning a splice junction unique to BimL.
This junction does not exist in BimEL, because of
the insertion of an additional coding exon,15
explaining
why mAb 5E5 does not bind this isoform. Because of their high degree of
specificity and their ability to work in many applications (Figure 2G)
,
mAbs 5E5 and 14A8 were chosen to determine Bim expression and
subcellular localization in cultured cell lines and normal mouse
tissues.
Expression of Bim in Cultured Cell Lines
Our initial attempts to detect Bim in cultured cell lines by Western blotting of lysates failed because of the low level of expression in these cells. Bim protein could be revealed, however, by immunoprecipitation of lysates from 107 cells followed by Western blotting (IP/Western). In many of the cell lines tested, mAb 5E5 detected the ~19-kd BimL protein, and in some cell lines mAb 14A8 revealed both BimL and the ~23-kd BimEL protein. BimS was never observed in any of these cell lines, indicating that this isoform is not normally expressed or is present at levels below the limits of detection.
Examples of IP/Western blots are shown in Figure 3
; the results are summarized in Table 1
.
The highest levels of BimL and
BimEL were found in the human breast
cancer-derived epithelial cell line MCF-7 and in several lymphoid
lines, such as the mouse T-lymphoid lines B6.2.16.BW2 and K052.DA20,
the human T-lymphoma-derived cell line Jurkat and the mouse
pre-B-lymphoma line ABLS8.1, and the mouse plasmacytoma lines SP2/0 and
NS1. Relatively low levels of BimL and
BimEL were detected in the mouse IL-3-dependent
myeloid line FDC-P1 and in the human kidney cell line 293T. Some cell
lines, such as the mouse macrophage line J774 and the monkey kidney
cell line Cos M6 only expressed BimL, and others,
such as the fibroblast cell lines NIH3T3, Rat1, and L929; the thymic
stromal line S17; and the erythroleukemia-derived lines FN4, DP16, and
Ts5 did not express any of the Bim isoforms. These studies
demonstrate that BimL and
BimEL are expressed in some cell lines, mainly
those of hematopoietic and epithelial origins.
IP/Western Analysis of Bim Expression in Mouse Tissues
IP/Western was also used to investigate the expression of Bim in
normal mouse tissues. In lymphoid tissues, such as spleen, thymus, and
lymph nodes, both mAbs 5E5 and 14A8 detected the
BimL protein and mAb 14A8 also recognized the
BimEL protein (Figure 4)
. Low levels of
BimL were observed in the pancreas, small
intestine, and stomach, and low levels of BimEL
could be seen in the testes. BimS was not
detected in any of the tissues analyzed, consistent with our analysis
of its expression in cell lines. The specificity of the mAbs was
confirmed by the observation that they immunoprecipitated Bim only from
tissues of normal but not Bim-deficient mice (Figure 3B)
. These results
demonstrate that BimL and
BimEL are expressed in cells from hematopoietic
tissues, in the gastrointestinal tract, and in reproductive organs.
Immunohistochemical Analysis of Bim Expression in Mouse Tissues
The expression pattern of the Bim protein in mouse tissues was investigated in detail by immunohistochemical staining, using mAbs 5E5 and 14A8. Because of the low levels of Bim we used the potent tyramide amplification system (see Materials and Methods), which increases the signal up to 1000-fold. Confirmatory results were obtained with two additional, independently derived anti-Bim mAbs (4E4 and 9F5), and as negative controls tissues were stained with isotype-matched rat mAbs. The specificity of the immunostaining was further validated by demonstrating that immunostaining by mAbs 5E5 and 14A8 could be blocked by preadsorption with recombinant GST-BimL. The results of Bim immunostaining were also confirmed by showing that they coincided perfectly with the pattern of in situ hybridization produced with a bim antisense probe.
Bim Expression in Hematopoietic Tissues
Among the hematopoietic cells of the bone marrow, megakaryocytes were intensely stained by both anti-Bim mAbs, and polymorphonuclear cells also had strong BimL/EL immunoreactivity. The intensity of BimL/EL immunoreactivity within the lymphocyte population was variable; some cells were stained strongly but others only weakly. Granulocytes had low but significant levels of Bim, whereas both erythroid precursors and mature erythroid cells were negative.
Examination of Bim expression in the thymus showed intense
BimL/EL immunoreactivity (Figure 5, A and B)
and bim mRNA
expression (Figure 5, C and D)
in the cortical thymocytes, where the
immature short-lived CD4+8+
pre-T cells are located. In contrast, expression of Bim protein and
bim mRNA was significantly lower (or absent) in the thymic
medulla, which contains the mature
CD4+8- and
CD4-8+ T cells. The only
cells in the medulla that stained weakly for Bim protein and
bim mRNA were large epithelial and macrophage-like cells.
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. Because of their
larger cytoplasm, immunohistochemical staining is probably more
prominent in activated, compared to quiescent, lymphocytes. The
paracortical areas and reticular cells in the medullary cords also
showed significant levels of Bim protein expression, although
this was more heterogeneous than that seen in primary follicles. In the spleen, BimL/EL immunoreactivity appeared to be limited to lymphoid cells in the white pulp, where staining was most intense in the germinal centers and the periarteriolar sheath/marginal zone. In the red pulp, scattered megakaryocytes and some macrophage-type cells exhibited moderate BimL/EL immunoreactivity, whereas erythrocytes did not. Bim mRNA expression in the spleen was limited to germinal center cells and a subset of cells in the white pulp (probably the T cell zone).
Bim Expression in the Central Nervous System
In the nervous system, immunostaining and in situ hybridization revealed Bim expression primarily in neurons in the gray matter, with no expression in glial, astrocytes, or oligodendrocytes or various types of supporting cells. Expression of BimEL was dominant in both intensity and distribution compared to BimL.
In the cerebellum, mAb 5E5 (anti-BimL) stained
within the granular layer, probably the Purkinje axons that extend into
this region, but not the stellate or granule cells. In contrast, mAb
14A8 (anti-BimS,L,EL) and in situ
hybridization with a bim antisense probe strikingly labeled
the Purkinje neurons and their associated dendrites projecting into the
molecular layer (Figure 6, A–D)
.
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. In the cerebral cortex varying numbers of small
pyramidal neurons and large motor neurons displayed a punctate
cytoplasmic distribution of Bim immunoreactivity (Figure 6, I–L)
. The
staining intensity varied according to the layer examined, and in
general many more neurons were labeled with mAb 14A8
(anti-BimS,L,EL) than with mAb 5E5
(anti-BimL). Intense
BimL/EL immunoreactivity was also observed in the
cuboidal cells of the choroid plexus. In situ hybridization
analysis of the brain confirmed the immunohistochemical analyses
(Figure 6)
. Bim Expression in the Gastrointestinal and Respiratory Systems
In addition to cells of hematopoietic and neuronal origin,
BimL/EL protein and bim mRNA were also
detected in several cells of epithelial origin within the digestive
tract. Moderate to strong BimL/EL
immunoreactivity and prominent signal from in situ
hybridization with a bim antisense probe were observed in
the serous secretory cells of the submandibular and parotid glands.
BimL protein expression extended to the columnar
or low cuboidal cells of the interlobular and intercalated ducts of the
submandibular, parotid, and sublingual glands (Figure 7, A–D)
.
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Moderate BimL/EL immunoreactivity was observed in the stratified squamous epithelia of the tongue (data not shown). In situ hybridization further revealed that the stratum granulosum and, to a lesser extent, the germinativum expressed bim mRNA, but the basal layer of the germinativum of the tongue did not. The serous secreting cells composing the tubuloalveolar glands (Von Ebner’s glands) of the vallate papillae in the tongue also had moderate to intense BimL or BimEL staining and bim mRNA expression.
The smooth muscle, the submucosa, and the serosa of the nonglandular portion of the stomach were marked by the absence of Bim immunoreactivity (with the minor exception of low-level staining of fibrocytes within the connective tissue) (data not shown). In contrast, Bim expression was readily apparent in the glandular portion of the stomach (fundus), where the parietal cells of the crypts, the outer epithelia, and neck cells all showed moderate BimL/EL immunoreactivity. The chief (zymogenic) cells did not express Bim. The simple columnar mucus epithelium of the pyloric region also showed moderate levels of BimL/EL immunoreactivity, but the pyloric glands did not. All of these observations were confirmed by in situ hybridization.
In the small intestine, high levels of BimL/EL
immunoreactivity were observed in the simple columnar epithelial cells
(enterocytes) of the villi and in crypt cells at the base of the villi
(Figure 8, A–D)
. Moderate levels of
BimL/EL expression were seen in the epithelial
cells of the crypts of the colon. The lymphoid cells of the
Peyer’s patches also showed moderate expression of
BimL/EL, but the epithelial goblet cells and the
lamina propria were negative.
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The cuboidal epithelium of the thyroid follicles had moderate levels of BimL immunoreactivity, and the chief (secretory) cells of the parathyroid gland expressed high levels of BimL/EL.
In the pancreas BimL/EL immunoreactivity was moderate in the acinar cells that are responsible for secretion of digestive enzymes, but stronger in the endocrine cells of the islets of Langerhans and the columnar epithelial cells lining the pancreatic ducts. In situ hybridization confirmed that bim expression in the pancreas was restricted to the exocrine acinar cells and that expression of Bim in the islet cells was nonspecific.
The cells of the adrenal gland displayed a heterogeneous pattern of Bim
protein and bim mRNA expression. The aldosterone-producing
cells of the outer cortical zona glomerulosa and the chromaffin cells
of the adrenal medulla both expressed moderate levels of
BimL and bim mRNA (Figure 7G)
, whereas
BimEL was found only in the adrenal medulla
(Figure 7E)
. No Bim immunoreactivity was found in the x-zone in virgin
female mice between the cortex and the medulla or in the
glucocorticoid-secreting cells of the zona fasiculata.
Bim Expression in Reproductive Organs
In the testes of adult male mice, intense
BimL immunostaining was observed in the cytoplasm
of elongating spermatids, becoming concentrated around the midpiece.
BimL expression was barely detectable in the
spermatogonia and spermatocytes (Figure 9A)
. In contrast,
BimEL expression could be demonstrated in
elongating spermatids as well as in Sertoli cells, whereas labeling of
spermatocytes was much less intense (Figure 9B)
. The ciliated columnar
epithelia of the epididymal ducts and the epithelial cells of the
ductus deferens displayed moderate BimL staining
and high-intensity BimEL immunolabeling. In
situ hybridization reproduced the staining seen with anti-Bim mAb
14A8 (Figure 9D)
.
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. The simple
columnar ciliated epithelium of the oviduct and the epithelial cells of
the uterus contained prominent BimL/EL
immunoreactivity.
In mammary glands of virgin female mice bim mRNA expression
was observed in the ductal and lobular cuboidal epithelial cells
(Figure 10A)
. During pregnancy
bim mRNA expression extended to the epithelial side
branching (Figure 10B)
, and this persisted throughout lactation (Figure 10C)
and involution (Figure 10D)
.
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BimL/EL expression was very striking in the hair follicles, the cylindrical down-growths of the surface epithelium. Intense labeling was seen in the undifferentiated bulb cells within the hair follicle (not shown), the outer epithelial sheath (which may represent a population of keratinocyte stem cells).36 No staining was observed in the connective tissue root sheath, and only weak labeling was detected in the sebaceous glands. Consistent with these results, high levels of bim mRNA expression were detected by in situ hybridization in hair follicles.
Bim Expression in Cardiovascular and Skeletal Muscles
Consistent with the IP/Western analysis (Figure 4)
, no Bim protein
was found in cardiomyocytes or skeletal muscle. Minimal Bim
immunolabeling was observed around the outer walls of the cardiac
chambers with mAb 5E5, but this was probably nonspecific, because no
immunostaining was observed with mAb 14A8 and no bim mRNA
could be revealed by in situ hybridization (data not shown).
Bim Expression in the Renal System
In the kidney, BimL/EL expression was strongest in the tubular epithelial cells of the proximal convoluted tubules at the corticomedullary junction. Only weak immunoreactivity was seen in the loops of Henle, the collecting ducts, and the distal convoluted tubules. Prominent bim mRNA expression was observed with certainty only in the ciliated epithelial cells constituting the proximal convoluted tubules within the renal cortex. No Bim immunoreactivity was detected in the glomeruli, indicating that Bim expression in the renal system was restricted to the ductal epithelial cells.
Collectively, these results demonstrate that BimL and BimEL are expressed in lymphocytes, myeloid cells, neurons, many different types of epithelial cells, and neuroendocrine hormone-secreting cells, and in somatic as well as germ cells of the reproductive organs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Gene targeting experiments in
mice have demonstrated that Bim is required for some but not all
apoptotic stimuli that can be inhibited by Bcl-2 or its relatives.
Within the whole animal, Bim has a critical role during embryonic
development, regulates hematopoietic cell homeostasis, and prevents
autoimmunity.17 Using immunohistochemical staining, IP/Western blotting, and in situ hybridization, we have defined the expression pattern of Bim. The two mAbs 5E5 and 14A8 were used to discriminate between the BimL and BimEL isoforms. No evidence for the expression of the third isoform, BimS, was obtained. This may indicate that BimS is not expressed or is produced only in a very small number of cells that have to be killed very rapidly.
The levels of BimL and BimEL are generally very low because both proteins can only be detected by extremely sensitive techniques. Interestingly, the absence of Bim provides a similar level of protection against cytokine withdrawal-induced apoptosis in lymphocytes17 as overexpression of Bcl-2.37,38 Thus Bim (and other BH3-only proteins) may not kill cells simply by inactivating pro-survival Bcl-2 family members in a simple one-to-one interaction. A speculative explanation is that anti-apoptotic Bcl-2 family members exist on intracellular membranes as a loosely aggregated macromolecular structure that regulates the activity of Apaf-1/CED-4-like adaptors. By binding to a small number of antiapoptotic Bcl-2 family members, BH3-only proteins may alter the overall structure of such a complex, thereby allowing caspase activation (see ref 39 ).
Some hematopoietic tissues that normally express
BimL and/or BimEL are
hyperplastic in Bim-deficient mice, demonstrating that this
pro-apoptotic protein is the cause of a nonredundant killing
activity in these cells.17
BimL and,
in most instances, BimEL are expressed in B- and
T-lymphoid cells, megakaryocytes, and myeloid cells (Figures 3–5
and
Table 1
). Both proteins are particularly prominent in cells with a high
turnover rate, such as cortical
CD4+8+ thymocytes, germinal
center B cells, and plasma cells. Consistent with this pattern of
expression, Bim-deficient mice have an accumulation of B cells, T
cells, monocytes, and granulocytes and perturbed T
lymphopoiesis.17
Although numbers of megakaryocytes are
normal in Bim-deficient mice, they have a 50% decrease in
platelets.17
Similar observations were made in mice
expressing a bcl-2 transgene in
megakaryocytes.55
This may indicate that platelet shedding
occurs by an apoptosis-like mechanism that is regulated by Bim and
perhaps by other members of the Bcl-2 family. Older bim-/-
mice had a 30–200-fold increase in IgG-secreting plasma cells and
elevated serum IgM, IgG, and IgA levels, and within a year most
succumbed to a systemic lupus erythematosus-like autoimmune
disease.17
Thus, although other BH3-only proteins and
Bax-related proapoptotic Bcl-2 family members are expressed in lymphoid
and myeloid cells,40-42
they are unable to compensate for
the absence of Bim. It is also possible that other pro-apoptotic Bcl-2
family members can act in conjunction with Bim, and it will therefore
be interesting to determine whether the absence of Bad, Blk, Bax, Bak,
or related proteins can exacerbate the lymphoid hyperplasia induced by
Bim deficiency.
The expression of Bim is not restricted to hematopoietic cells, but can also be detected by immunohistochemistry and by in situ hybridization in a wide variety of cells of epithelial origin. Epithelia constitute a diverse group of tissues that are involved in a wide range of activities such as absorption, secretion by glandular structures (invaginations of epithelial surfaces), and protection against invading pathogens. As in the case of Bim, the epithelia of many tissues, such as renal tubules, small intestine, breast, prostate, and respiratory tract, also express Bax,40 Bak,41 and possibly other BH3-only proteins. Many epithelial cells also express anti-apoptotic proteins, including Bcl-2,40 Bcl-xL,43 and Mcl-1.44 Certain complex epithelia have a striking gradient of expression of pro- versus anti-apoptotic Bcl-2 family members. For example, Bcl-2 was predominantly located in the less differentiated cells lining the epithelial basement membrane, whereas Bax and Mcl-1 were prominent in the more differentiated short-lived cells located in the upper layers of epithelial tissue. Notably, expression of Bim and Bax at the base of the crypts of the small intestinal mucosa is consistent with the reports of high rates of spontaneous and genotoxic damage-inducible apoptosis in this region.45 Bim expression in the hair follicle bulb is restricted to precursor cells, which, under the inductive influence of the dermal papilla, undergo a highly ordered process of cell death that is critical for hair production.46 Interestingly, transgenic mice that overexpress Bcl-xL in keratinocytes under the keratin 14 promoter have abnormally short hair,47 and Bcl-2-deficient mice become prematurely gray.48 Thus both pro- and anti-apoptotic proteins appear to have a regulatory role in hair production.
So far we have not found any abnormalities in epithelial cells in Bim-deficient mice,17 and this might be due to the functional overlap between pro-apoptotic Bcl-2 family members. However, the specific function of Bim may only be revealed when epithelia are subjected to mechanical stress or other death-promoting insults.
BimL and, in particular, BimEL are expressed in neuronal cells of the central nervous system (CNS). Indeed, like Bim, Bax has been found in several populations of long-lived neurons, including the Purkinje neurons of the cerebellum and large neurons of the cortex.40 Expression of such proapoptotic proteins coupled with low Bcl-2 levels may explain why these neurons are very sensitive to induction of apoptosis by neurotrophin withdrawal, hypoxia, and hypoglycemia.49 It is also possible that BH3-only proteins are involved in the attrition of neurons that occurs normally during development of the mammalian nervous system.50 Although Bim-deficient mice have no obvious defects in the CNS,17 under conditions of neuronal stress abnormalities may become apparent. It is also likely that other BH3-only proteins can compensate for Bim in the nervous system. Interestingly, expression of a related protein, Hrk/DP5, is dramatically increased in cultured neurons when they are deprived of neurotrophin.51 It is therefore possible that this protein is the dominant proapoptotic BH3-only protein in neurons. Alternatively, the function of this subfamily in neurons may only become apparent in mice lacking more than one of these proteins.
BimL and BimEL are both
expressed in somatic as well as germ cells of female and male
reproductive organs (Figures 4 and 9)
. Despite this, Bim-deficient mice
from both sexes have normal fertility.17
This may be
explained by the expression of many other proapoptotic Bcl-2 family
members, such as Bax, Bak, Bok/Mtd, and Bad, in these
tissues.40,41,52
These molecules may have overlapping
functions. It is clear, however, that Bcl-2 family members are
essential for spermatogenesis, because Bax-53
and
Bcl-w-deficient male mice32,54
are both infertile because
of defective spermatogenesis.
Approximately 65% of Bim-deficient embryos die during development before E9.5.17 The reason for this premature death remains unknown, but it is interesting to consider this in the context of the sites of Bim expression. Because these bim-/- embryos die so early, this is unlikely to be due to defects in neuronal cells or hematopoietic cells. Abnormalities in epithelial tissues, however, may provide an explanation. In an attempt to identify the essential function of Bim in early development, we are determining Bim expression during embryogenesis and are trying to pinpoint the developmental stage in which Bim plays such an essential role.
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Acknowledgements |
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Footnotes |
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Supported by grants and fellowships from AMRAD Biotech, the Leukemia and Lymphoma Society of America (New York), the Anti-Cancer Council of Victoria (Melbourne, Australia), the Dr. Josef Steiner Cancer Research Foundation (Bern, Switzerland), the National Health and Medical Research Council (Canberra, Australia), the Cancer Research Institute (New York), and the Victorian Breast Cancer Research Consortium (Melbourne, Australia).
Dr. Print’s present address is Department of Pathology, University of Cambridge, Cambridge, UK.
Accepted for publication April 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ntgen F, Adams JM, Strasser A: Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 1999, 286:1735-1738
-Cell neogenesis in an animal model of IDDM. Diabetes 1997, 46:599-606[Abstract]
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