The LIM Domain Protein Lmo4 Is Highly Expressed in Proliferating Mouse Epithelial Tissues
The Walter and Eliza Hall Institute of Medical Research and Bone Marrow Research Laboratories, Parkville, Australia
Correspondence to: Jane E. Visvader, PhD, VBCRC Laboratory, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3050, Australia. E-mail: visvader{at}wehi.edu.au
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Summary |
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(J Histochem Cytochem 53:475486, 2005)
Key Words: LMO4 monoclonal antibody immunohistochemistry proliferation mouse organs
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Introduction |
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Targeted deletion of Lmo4 in mice results in perinatal lethality and complex phenotypic abnormalities. Approximately 50% of Lmo4 nullizygous mice exhibit exencephaly due to failure of the neural tube to close (Hahm et al. 2004; Tse et al. 2004
), whereas all mice show sphenoid bone defects (Hahm et al. 2004
). Homeotic transformations were evident in Lmo4-null embryos and newborn mice, including cranial nerve abnormalities and skeletal defects in the rib cage and cervical vertebrae (Hahm et al. 2004
). These transformations are reminiscent of defects that occur in Hox-deficient mice (Trainor and Krumlauf 2000
), suggesting that Lmo4 regulates the expression or activity of Hox proteins in specific cell types.
The importance of LMO proteins in regulating normal development is underscored by the findings that deregulated expression of these proteins can be oncogenic. The LMO1 and LMO2 genes were discovered by their translocation in acute T-cell leukemia and overexpression of either gene in transgenic mice results in T-cell tumors (Rabbitts 1998). Furthermore, two SCID (Severe Combined ImmunoDeficiency) patients undergoing gene therapy developed T-cell leukemia due to the ectopic activation of the LMO2 gene by retroviral integration (Hacein-Bey-Abina et al. 2003
). LMO4 was originally identified in an expression screen using autologous serum from a breast cancer patient (Racevskis et al. 1999
), and we have demonstrated that elevated levels of LMO4 occur in greater than 50% of primary breast tumors (Visvader et al. 2001
). The mechanism underlying LMO4 overexpression in breast cancer reflects increased promoter activity in addition to aberrant activation of a second promoter (Wittlin et al. 2003
). Somatic mutations in the LMO4 gene occur infrequently in breast cancers (Sutherland et al. 2003
). Increased levels of LMO4 have also been reported in cancers of the oral cavity, in which high levels were apparent in less-differentiated tumors (Mizunuma et al. 2003
).
In contrast to the more restricted expression profiles of the other LMO genes, Lmo4 mRNA is widely expressed in mouse embryonic and adult tissues (Grutz et al. 1998; Kenny et al. 1998
; Sugihara et al. 1998
; Racevskis et al. 1999
; Bulchand et al. 2003
). Interestingly, the pattern of Lmo4 expression during embryogenesis defined uncommitted cells, indicating that one function of Lmo4 may be to inhibit differentiation (Kenny et al. 1998
). To perform a comprehensive analysis of Lmo4 expression in adult mouse organs at the protein level, we generated monoclonal antibodies (MAbs) specific for Lmo4. Immunohistochemical analysis revealed that although Lmo4 is widely distributed, its expression is restricted to specific cell subtypes. Lmo4 protein was found to be particularly prevalent in epithelial tissues, with high levels occurring in proliferating cells. Our results imply a role for Lmo4 in regulating proliferation in different epithelia.
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Materials and Methods |
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Expression Constructs and Protein Purification
Full-length mouse Lmo4 cDNA was amplified by PCR using forward 5'-CGC GGA TCC AGC TCG CAG CCG CCC CCG GTG-3' and reverse 5'-CGC GGA TCC TCA GCA GAC CTT CTG GTC TGC CAG-3' primers, then subcloned into the BamHI site of pGEX-2T (Amersham Biosciences, Inc.; Uppsala, Sweden). The production of GST-Lmo4 protein and subsequent purification on a glutathioneSepharose column (Amersham Biosciences, Inc.) was performed according to standard protocols. The expression constructs encoding either FLAG-tagged full-length LIM domain 1 (LIM1) or LIM domain 2 (LIM2) of mouse Lmo4 have been described (Sum et al. 2002).
Immunization and Hybridoma Fusion
Wistar rats were initially immunized by subcutaneous (SC) injection of 100 µg of purified GST-Lmo4 protein dissolved in complete Freund's adjuvant (Difco; Detroit, MI). Two subsequent boosts of the immunogen were injected SC 3 and 6 weeks later. A final boost of protein dissolved in phosphate-buffered saline (PBS) was given by intravenous and intraperitoneal injection 4 weeks later. Hybridomas were generated 3 days after the final boost by fusing spleen cells from immunized rats with the SP2/0 myeloma cell line (Galfre et al. 1977; O'Reilly et al. 1998
). Hybridomas producing MAbs to Lmo4 were identified and their isotype determined by a FACScan screening strategy that we have previously described (O'Reilly et al. 1998
; O'Reilly et al. 2002
). Briefly, 293T cells were transiently transfected with an expression construct containing mouse Lmo4 cDNA with an N-terminal FLAG tag. Transfected cells were fixed in 1% paraformaldehyde, permeabilized with 0.3% saponin (Sigma), and stained with hybridoma supernatants. Bound antibodies were detected by fluoresceinisothiocyanate (FITC)-conjugated goat anti-rat Ig antibodies (Southern Biotechnology Associates, Inc.; Birmingham, AL) and analyzed in a FACScan analyzer (Becton Dickinson: Bedford, MA). Hybridomas producing Lmo4-specific antibodies were cloned twice and subsequently 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; Asheville, NC). Antibodies were purified on a protein GSepharose column (Amersham Biosciences, Inc.) according to the manufacturer's protocols.
Flow Cytometry
Balb/c mouse-derived thymocytes were incubated with a mixture of PE-conjugated CD4+ (BD Pharmingen; San Diego, CA) and FITC-conjugated CD8+-specific MAbs (kindly provided by A. Strasser) for 30 min at 4C. The immunostained thymocytes were subjected to a four-way-sort on a FACS sorter (DIVA; Becton Dickinson) after gating for PI-positive cells to isolate each of the four thymic subsets: CD4CD8 double negative cells, CD4+CD8+ double-positive cells, and single-positive CD4+ and CD8+ cells. Protein lysates (from 105 cells) were subjected to Western blot analysis.
Western Blotting
Protein lysates from wild-type and Lmo4-deficient mouse E16.5 embryos were prepared by crushing frozen embryos in liquid nitrogen with a mortar and pestle before transfer into 1 ml ice-cold lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris/HCl (pH 7.5), 1% NP40, 1 mM DTT, supplemented with Complete protease inhibitor tablet (Roche Diagnostics; Mannheim, Germany), 10 mM NaF, and 1 mM Na3VO4). For the preparation of tissue lysates, frozen tissues derived from adult mouse organs were pulverized in liquid nitrogen before transfer into ice-cold ONYX lysis buffer (20 mM Tris-HCl (pH 7.4), 135 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, and 1% Triton X-100; supplemented with Complete inhibitor tablet, 50 mM NaF, and 1 mM Na3VO4) or directly homogenized in ONYX (ONYX Pharmaceuticals; Richmond, CA) lysis buffer at 4C as detailed elsewhere (Print et al. 1998).
Tissue lysates (3050 µg total protein) for Western blot analysis were denatured by boiling in sodium dodecyl sulfate (SDS) loading buffer and separated on polyacrylamide gels (Novex; Carlsbad, CA) before transfer to polyvinylidene difluoride membranes (Millipore; Bedford, MA). Nonspecific binding of proteins to membranes was blocked by incubation in PBS containing 5% skim milk and 0.1% Tween-20. The membranes were then probed with anti-Lmo4 MAbs (12 µg/ml), followed by horseradish peroxidase-coupled secondary antibodies (Amersham Biosciences, Inc.), and developed by ECL (Amersham Biosciences, Inc.). To control for the integrity of proteins in cell and tissue lysates, the blots were probed with mouse anti-Hsp70 N6 (kindly provided by Dr R. Anderson, Peter MacCallum Cancer Centre, Melbourne, Australia and Dr. W. Welch, UCSF, San Francisco, CA), anti-tubulin (Sigma; St. Louis, MO), or anti-actin (Sigma) MAbs.
Immunohistochemistry
For immunohistochemistry, mouse tissues were fixed in 4% paraformaldehyde. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval by boiling in 10 mM citrate buffer (pH 6.0) for 20 min. After cooling to room temperature (RT), sections were treated with 3% hydrogen peroxide to block endogenous peroxide activity, followed by a blocking step in PBS containing 0.2% Triton X-100 (T-PBS) and 10% normal rabbit serum (NRS). Sections were then incubated overnight at 4C with rat anti-Lmo4 20F8 or 16H2 (510 µg/ml), rat anti-mouse Ki-67 (1:50 dilution; DAKO) or an isotype-matched control rat antibody (IgG2a/, Pharmingen) diluted in T-PBS containing 5% NRS. Staining was detected by incubating with biotinylated rabbit anti-rat IgG antibody (DAKO), followed by HRP-conjugated strepavidin (LSAB2, DAKO). Finally, sections were stained with diaminobenzidene (DAKO), counterstained with hematoxylin, and mounted in DPX (BDH; Poole, UK).
Wholemount Staining for ß-Galactosidase Activity
Tissues from Lmo4:LacZ KI mice were fixed in PBS containing 2% paraformaldehyde and 0.25% glutaraldehyde for 2 hr at 4C prior to staining for ß-galactosidase activity by incubation with X-gal solution (5 mM K3Fe, 5 mM K4Fe, 2 mM MgCl2, and 0.02% NP40 in PBS, supplemented with 0.5 mg/ml X-gal) at 37C overnight. These tissues were then postfixed in 4% paraformaldehyde overnight at 4C and then embedded in paraffin. Sections (7 µm) were prepared and counterstained with nuclear fast red.
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Results |
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Immunohistochemical Analysis of Lmo4 Expression in Mouse Tissues
The Developing and Adult Central Nervous System
The specificity of Lmo4 immunostaining by 20F8 MAb was validated using sections from wild-type and Lmo4-deficient E16.5 mouse embryos (Figures 2A2D). As expected, no immunostaining was detectable in Lmo4-deficient embryos (Figures 2B and 2D), whereas strong staining was evident in wild-type tissue (Figures 2A and 2C). Approximately 50% of these embryos exhibited exencephaly (Hahm et al. 2004), as evident in Figure 2D. The immunohistochemical profile of Lmo4 was confirmed using a second anti-Lmo4 MAb, 16H2 (data not shown). In addition, an isotype-matched rat non-immune IgG provided a negative control for each experiment (Figure 2, insets).
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In the adult central nervous system (CNS), abundant Lmo4 expression was observed in neurons and astrocytes in the hippocampus (Figures 2E and 2G) and cerebral cortex (data not shown). Lmo4 primarily localized to the nuclei in these cells, with little or no staining of the cytoplasm. Conversely, the larger motor neurons and Purkinje cells (Figure 2H) exhibited high levels of cytoplasmic Lmo4 protein. Within chick embryos, Lmo4 has been localized to both muscle sensory and motor neurons (Chen et al. 2002). Lmo4 was also detected in the choroid plexus and ependymal cell layers, which are derived from neuroepithelium (Figure 2F), but no Lmo4 protein was detected in oligodendrocytes (Figures 2E2G). This expression profile is consistent with that described for LMO4 in both mouse and human brain (Hermanson et al. 1999
; Leuba et al. 2004
).
Lmo4 Expression in Various Mouse Organs
The widespread expression of Lmo4 determined by Western blot analysis (Figure 1B) was further characterized by immunohistochemistry using the MAb 20F8 (Figures 35 and Table 2). The expression profile of Lmo4 was also confirmed by staining tissue sections from Lmo4:LacZ KI mice, in which the lacZ gene is driven by the endogenous Lmo4 promoter (Hahm et al. 2004) for ß-galactosidase activity (Figures 35). An identical pattern of expression was observed in all cases. In addition, staining with an isotype-matched rat non-immune IgG provided a negative control for each tissue (Figures 35).
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Respiratory Tract
Lmo4 expression was confined to the low columnar epithelial cells lining the trachea and lumen of the respiratory bronchioles and alveoli in the lung (Figures 3D and 3E). Lmo4 immunostaining was also present in the non-ciliated Clara cells, identified by a characteristic bulging into the lumen, as well as in the terminally differentiated ciliated cells (Figure 3E).
Pancreas
Intense Lmo4 immunostaining was detected in the endocrine islet of Langerhans cells but was negligible in the exocrine acinar cells (Figure 3H), consistent with the expression of LacZ in pancreatic tissue from LMO4:LacZ KI mice (Figure 3G). A lower level of Lmo4 protein expression was also detected in epithelial cells lining the pancreatic ducts.
Kidney
Lmo4 expression was readily detectable in all tubular structures of the kidney, including the proximal and distal convoluted tubules, by both LacZ staining and immunohistochemistry using 20F8 MAb (Figures 3J and 3K). Stronger staining of juxtaglomerular tubules was observed (Figure 3K), whereas the glomeruli were devoid of Lmo4 expression. The majority of cells in the kidney expressed relatively low levels of Lmo4, consistent with the level observed by Western analysis (Figure 1B).
Lmo4 Is Highly Expressed in Proliferating Cells of Epithelial Tissues
Skin and Hair Follicles
Lmo4 was predominantly expressed in hair follicles (Figures 4A and 4B) and squamous epithelial cells in the basal layer of the epidermis (Figures 4A, 4B, and 5B). Within the hair follicle, Lmo4 immunostaining was evident in the sebaceous glands, undifferentiated bulb cells, and the outer epithelial root sheath (Figure 4B), the latter of which may contain a keratinocyte stem cell population (Rochat et al. 1994). Epidermal basal cells have abundant Lmo4 protein and are highly proliferative as indicated by the expression of the proliferation marker Ki-67 (Figure 5A).
Tongue
Lmo4 expression was evident in the stratified squamous epithelial cells of the tongue, with the majority of expression confined to the basal layer of the germinativum (Figures 4D and 4E). Nuclear Lmo4 expression was also detected in taste buds and serous glands within the tongue (Figure 4E), but no expression was evident in the filiform or dermal papilla.
Reproductive Organs
Lmo4 was expressed in both the uterine myometrium and endometrium in the female reproductive system (Figures 4G and 4H). Strong cytoplasmic immunostaining was observed in the endometrial cells lining the uterine glands and the lumen of the uterus (Figure 4H), both of which are highly proliferative and turn over regularly during the estrus cycle. Expression of Lmo4 in the ovary was less striking, with low to moderate staining of the corpus luteal and follicular cells and negligible staining of the single epithelial layer (Table 2). In male reproductive organs, Lmo4 expression was confined to spermatogonium and spermatocytes in the testes, whereas low levels were evident in the epididymis and epithelial cells lining the dorsal and ventral prostate (Table 2).
Mammary Gland
The mid-pregnant mammary gland exhibited prominent Lmo4 expression in both the ductal and lobuloalveolar epithelial cells, with substantially lower levels in the myoepithelium and surrounding stroma (Figures 4J and 4K). Lmo4 expression was also detected within the terminal end bud (TEB) of the peri-pubertal mammary gland (Figure 5E). Intense Lmo4 staining was observed in the cap cells, which form the outer monolayer at the tip of the bulbous structure (Figure 5E). These are highly proliferating cells, as shown by their expression of the Ki-67 antigen (Figure 5D).
Small Intestine
Strong Lmo4 expression was detected in the crypts of Lieberkühn located at the base of villi (Figure 5H). These epithelial crypt cells also express abundant Ki-67 (Figure 5G), indicating a high proliferation rate. In addition to crypt cells, nuclear Lmo4 immunostaining was detected in the smooth muscle surrounding the small intestine (Figure 5H). Brunner's glands within the duodenum, representing modified extensions of crypt cells, also expressed moderate levels of Lmo4 (Table 2).
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Discussion |
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High levels of Lmo4 protein occur in epithelial tissues and in cells that appear to have a high proliferative capacity. The epithelial tissues include lung, kidney, skin, tongue, small intestine, uterus, and mammary gland. In these tissues, Lmo4 was localized primarily to the nucleus or distributed throughout both the nucleus and cytoplasm. Within several of the epithelial tissues, high levels of Lmo4 were observed in proliferating cells, particularly in their nuclei. Stratified squamous epithelial cells of the tongue and uterine endometrium exhibit rapid turnover and express abundant Lmo4. Mitotically active cells in the basal layer of the epidermis and undifferentiated cells in the outer root sheath of the hair follicle, thought to contain a stem cell population (Rochat et al. 1994), exhibited strong nuclear staining of Lmo4. Similarly, crypt cells at the base of the villi in the small intestine and cap cells of TEBs in the peri-pubertal mammary gland showed prominent Lmo4 expression. These cells have a high proliferative index as indicated by staining for the Ki-67 antigen. In the mammary gland, both ductal and lobuloalveolar epithelial cells expressed abundant Lmo4. The formation and expansion of the lobuloalveolar units during pregnancy is accompanied by a high rate of proliferation.
Lmo4 expression is not restricted to proliferating cells. High Lmo4 protein levels were detected in postmitotic and migrating neurons of the developing brain and in several different neuronal cell types within the adult CNS, including astrocytes, motor neurons, and Purkinje cells. Interestingly, a high level of Lmo4 expression has been reported in the entorhinal cortex and CA1 hippocampal region of the human brain, which are altered in Alzheimer's disease (Leuba et al. 2004). Comparison of Lmo4 expression in these regions revealed reduced levels in brains from Alzheimer's disease patients relative to control brains (Leuba et al. 2004
). Lmo4 has been found to be differentially expressed at an early stage in the development of muscle sensory neurons and may actively participate in their differentiation by regulating the transcriptional activity of LIM homeodomain proteins (Chen et al. 2002
). In several other tissues, such as smooth muscle, kidney and pancreas, mature cells were also found to express abundant Lmo4. Thus, Lmo4 may have a dual physiological role in which it can either promote proliferation or differentiation.
Lmo4 is widely expressed in both embryonic and adult mouse tissues but in restricted cell types and at levels that vary markedly. The expression profile of Lmo4 suggests that this protein is multifunctional and that it influences both proliferation and differentiation, dependent on cellular context. The underlying cause of death in Lmo4 null mice, all of which succumb within a few minutes of birth, is yet to be established. Histological examination of major organs, including the hematopoietic compartment, did not reveal any gross morphological defects in these mice (Hahm et al. 2004; Tse et al. 2004
). On the basis of high Lmo4 expression in the lungs and brain, it is tempting to speculate that proper lung expansion or brain stem function may be affected in these mutants, leading to perinatal lethality. Specific deletion of the Lmo4 locus in lung or specific regions of the brain should provide insight into the physiological role of Lmo4 in these organs.
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Acknowledgments |
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We wish to thank F. Feleppa and U. Devisekera for expert technical assistance and S. Mihajlovic for histology.
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Footnotes |
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