Smooth Muscle-specific Tropomyosin Is a Marker of Fully Differentiated Smooth Muscle in Lung
Oncology Research Unit (BV,GS,PWG,RPW) and Department of Respiratory Medicine (KM), The Children's Hospital at Westmead, NSW, Australia, and Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, Australia (KM,GS,PWG,RPW)
Correspondence to: Prof. Peter Gunning, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia. E-mail: peterg3{at}chw.edu.au
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Summary |
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Key Words: actin antibody lung smooth muscle tropomyosin
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Introduction |
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Tropomyosins (Tm) are rod-like helical proteins that dimerize and bind to actin. In smooth muscle cells, Tm is likely to play a role in the stabilization of the smooth muscle actin contractile filaments, similar to its role in non-muscle cells. The four Tm genes produce many isoforms (>40) as a result of alternative exon usage. Most are found in non-muscle cells, but there are some specific to either striated or smooth muscle. Only two isoforms appear to be specific to smooth muscleone from the ß Tm gene and the other from the Tm gene.
-sm Tm contains a unique exon 2a, not found in any other Tm, whereas ß-sm Tm uses the same 2b exon as muscle isoforms (Figure 1). No specific antibody exists to either of the two smooth muscle isoforms of Tm, although other antibodies can detect them both but these also detect other non-muscle or striated muscle isoforms, making them unable to be used to label smooth muscle specifically. Tropomyosin has not previously been widely studied in smooth muscle when compared with actin and myosin, partly due to a lack of specific antibodies (Owens 1995
).
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Materials and Methods |
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CG3 monoclonal antibody used at 1/500 dilution recognizes an epitope in the 1b exon of the Tm (TPM3) gene (Novy et al. 1993
) and was a gift from Dr. Jim Lin (University of Iowa; Iowa City, Iowa).
-sm Actin antibody (Sigma; St Louis, MO) was used at 1/500 dilution. Horseradish peroxidase (HRP)-conjugated donkey anti-mouse (Amersham; Buckinghamshire, UK) and donkey anti-sheep (Jackson ImmunoResearch; West Grove, PA) were used at 1/10,000. Alkaline phosphatase-conjugated secondaries (donkey anti-sheep and goat anti-mouse) were from Jackson ImmunoResearch and were used at 1/1000.
Tissue Preparation for Western Blotting
Whole tissues were removed from wild-type FVB strains of adult mice and immediately frozen in liquid nitrogen. Stomach and intestine were opened and rinsed with phosphate-buffered saline (PBS) prior to freezing. Tissues were homogenized in 50 mM Tris-Cl, pH 8.0, in a volume adequate to cover the tissue, using a Polytron blender. Samples of whole tissue homogenate were diluted half in solubilization buffer (100 mM Tris-Cl, pH 7.6, 2% SDS, 2 mM DTT) and then quantified using a BCA protein assay kit (Pierce; Rockford, IL). Twenty-µg samples were analyzed by 15% SDS-PAGE.
Tissue Preparation for Immunohistochemistry
Embryos from various stages of development and whole tissues from adult mouse were removed and fixed in 10% formalin before embedding in paraffin wax. Lungs from 1- and 7-day-old postnatal and adult mice were perfused with 10% formalin to inflate the airways by gravity flow from a height of 20 cm by cannulation of the trachea. The trachea was then tied off with suture cotton and the inflated lungs placed in 10% neutral-buffered formalin for 1 week before embedding in paraffin. Tissue sections were cut at 5 µm and placed onto a poly-L-lysine-coated slide (Menzel-Glaser; Braunscheig, Germany). Consecutive sections were used for staining.
Human lung tissue was obtained at coronial postmortem. Ethics approval was obtained from the Children's Hospital at Westmead Ethics Committee and written consent for use in research was obtained from the next of kin. Tissues were obtained from 4-month-old, 2-year-old, and 9-year-old females. The causes of death were unrelated to lung disease, although the 9-year-old female showed signs of inflammation and smooth muscle thickening consistent with undiagnosed, untreated chronic asthma. The other two samples showed no signs of lung pathology. The tissues were fixed by immersion in 10% neutral-buffered formalin and random samples were processed into paraffin blocks and sectioned onto slides. Several consecutive sections were used for staining. Human tissue for Western blotting was treated essentially as described for the mouse tissues.
Immunohistochemistry
Slides were dewaxed in xylene/ethanol, washed in PBS, and blocked in 10% fetal calf serum for 10 min. Primary antibody diluted in PBS was applied for 3 hr. Slides were washed in PBS and secondary antibody applied at 1/1000 dilution for 1 hr. Immunoreactivity was visualized by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics; Sydney, Australia) for 45 min. Sections were counterstained with nuclear fast red and dehydrated in ethanol/xylene before coverslipping.
Western Analysis
Gels were blotted at 80 V for 2 hr onto PVDF (Millipore; Bedford, MA) and blocked overnight at 4C in 5% skim milk powder. Blots were washed in Tris-buffered saline (100 mM Tris, pH 7.5, 150 mM NaCl; TBS) for 5 min and primary antibody applied in TBS for 2 hr with gentle agitation. Blots were washed in TBS/0.5% Tween 20 (TTBS) four times for 15 min each. HRP-conjugated secondary antibody was applied for 1 hr, followed by four 20-min washes with TTBS. Western lightning chemiluminescent reagent (Perkin Elmer Life Sciences; Norwalk, CT) was used according to manufacturer's instructions. Signal was visualized on X-ray film after exposures of between 2 and 60 min.
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Results |
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To characterize the -sm Tm isoform antibody, its expression and tissue distribution was analyzed by Western blotting. Figure 2A shows adult mouse tissues incubated with our
/2a Tm antibody. The antibody detected a band of expected size of 40 kDa in tissues known to have high levels of smooth muscle, such as lung, intestine, stomach, and aorta, as well as significant amounts in kidney, and on a much longer exposure a faint band could also be detected in the heart (data not shown) as predicted from the RT-PCR results of Muthuchamy et al. (1993)
. Longer exposures did not, however, reveal the presence of the 34-kDa
/2a containing Tm reported by Zajdel et al. (2002)
and Denz et al. (2004)
in axolotl and human hearts suggesting that if expressed in mouse it is below the level of detection with this antibody. An extra band of larger size was seen in the aorta. When these were incubated in the presence of the
/2a peptide, all 40-kDa bands were absent but this larger band in aorta was still present, indicating it is probably a nonspecific background band. Some degradation of actin was apparent in the stomach samples but this was not seen with Tm. When compared with the
-sm actin antibody, the pattern is similar. It therefore appears that both
-sm actin and
-sm Tm are coexpressed in the same tissues, but the relative level of expression of
-sm Tm between organs differs to that seen with
-sm actin, especially for the aorta, suggesting that the stoichiometry of
-sm Tm to
-sm actin may be different. Staining with CG3, which detects non-muscle isoforms from the
Tm gene, is shown as a control (Figure 2A). The expression is quite different and more widespread than for
/2a. The
/2a antibody also recognizes the same protein in human lung. Figure 2B shows reactivity of this antibody with a 39-kDa protein in human lung. The small difference in mobility between mouse and human may reflect a conformational difference based on five amino acid differences.
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Discussion |
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In all tissues examined, only one band was detected that was completely inhibited by addition of peptide as a competitor for the antibody. The staining pattern on histological samples could similarly be inhibited by the peptide (not shown). In organs such as brain and liver, the only positive staining detectable by immunohistochemistry was in blood vessels (not shown) with extended incubations only resulting in much background nonspecific staining. In contrast, our antibody shows a clear preference for smooth muscle structures in lung, gut, stomach, and esophagus, colocalizing with smooth muscle actin. The tight coexpression of -sm Tm and actin is consistent with the possibility that these isoforms may have a preferred association in the microfilaments of smooth muscle cells. We conclude that the
/2a antibody specifically recognizes a single form of tropomyosin found in smooth muscle and that it will be particularly useful in investigations of smooth muscle biology and pathophysiology.
Tropomyosin in the Lung
The developmental process in lung is well studied, so we chose to use lung as our model for -sm Tm expression. It is clear that in the mouse
-sm Tm is a protein that appears later in development than
-sm actin. Whereas
-sm actin appears at a high level from the earliest postnatal samples,
-sm Tm was just detectable at younger embryonic ages but increases in adult lung as shown by Western blotting. From immunohistochemical data,
-sm Tm does not appear to be expressed in significant quantities in the lung for protein detection until embryonic day 15.5, although mRNA is reported as detectable in embryonic stem cells and embryoid bodies (Muthuchamy et al. 1993
). We were unable to detect expression in skeletal muscle or cardiac muscle at any time.
-sm Tm protein was detected only in smooth muscle. The onset of expression appears to depend on tissue type, with intestine expressing early in development and lung quite late. This fits with the normal pattern of development because the lung is the last organ to mature prenatally and is embryologically derived from the gut. Indeed, the lung continues to develop postnatally and is not structurally mature until somewhere between the third and eighth year of life in humans. Results from the mouse correlate well with human, insofar as very little
-sm Tm is seen in human lung samples from a 4-month-old child. Interestingly, there were differences in onset of expression for lung airway smooth muscle and lung vascular smooth muscle. Other markers of smooth muscle show these differences also, such as calponin and actin, where expression is earlier in systemic blood vessels than pulmonary (Jostarndt-Fogen et al. 1998
).
-sm Actin is the earliest known marker of smooth-muscle-cell differentiation (Mitchell et al. 1990
; McHugh 1995
) but is not necessarily the most specific, because
-sm actin is transiently expressed in both cardiac and skeletal muscle (Woodcock-Mitchell et al. 1988
; McHugh 1995
). As early as 10 weeks gestation, human lung shows
-sm actin positive staining (Leslie et al. 1990
). Other markers of smooth muscle such as myosin heavy chain, vimentin, calponin, caldesmon, and desmin exist (Mitchell et al. 1990
; Halayko et al. 1996
; Low and White 1998
). A number of different isoforms of smooth muscle myosin heavy chain exist and, interestingly, these can be found in different cellular regions. For example, larger elastic arteries do not contain the smooth myosin-B isoform but airways and small blood vessels do (Low et al. 1999
). It may be that
-sm Tm is preferentially associated with a particular myosin isoform, because some heterogeneity is also noted with
-sm Tm staining. Indeed, a recent study has demonstrated that manipulation of Tm isoform composition of neuroepithelial cells can alter myosin recruitment to actin filaments (Bryce et al. 2003
). In studies thus far, ß-sm Tm and
-sm Tm are thought to be present in approximately equal amounts (Fatigati and Murphy 1984
), and it is thought that they dimerize. However, it has not been demonstrated that the two isoforms colocalize in all tissues. A
-smooth muscle isoform of actin also exists as well as the
-smooth muscle isoform and there may be differences in the distribution of
- and ß-sm Tm and their correlation with these actin isoforms. This may explain the difference in intensity we found with
-sm actin and
-sm Tm in aorta when compared with the other tissues. Using the existing nonspecific antibodies with
/2a may help shed light on isoform distribution and whether any switching occurs during development, analogous to that seen in brain development (Weinberger et al. 1996
; Schevzov et al. 1997
; Hannan et al. 1998
). Indeed, there is a growing body of evidence to suggest that many different cell types sort tropomyosin isoforms into spatially segregated actin populations in brain (Gunning et al. 1998a
,b
), fibroblasts (Percival et al. 2000
,2004
), epithelial cells (Dalby-Payne et al. 2003
), and skeletal muscle (Kee et al. 2004
).
Smooth muscle myosin heavy chain is highly specific for the smooth muscle lineage in development (Miano et al. 1994; Owens 1995
; Jostarndt-Fogen et al. 1998
; Ratajska et al. 2001
), but due to its later appearance
-sm Tm may be a better marker for fully differentiated smooth muscle especially in adult tissues and may be used to assess the degree of differentiation of vascular smooth muscle. The use of Tm as a marker for smooth muscle has not been possible until now due to a lack of specific antibodies. The
/2a antibody colocalizes with
-sm actin and can be used as a marker for differentiated smooth muscle in both human and mouse tissues. It may therefore potentially be useful for analysis of pathological states involving smooth muscle, such as in asthma where smooth muscle hypertrophy occurs, as well as in monitoring smooth muscle cell phenotypic modulation in cultured cells.
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Acknowledgments |
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We thank Ms. Janelle Mercieca for technical assistance with preparation and staining of histological samples. P.G. is a Principal Research Fellow of the NHMRC.
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Footnotes |
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