1 Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202; and 2 Department of Anatomy, University of Wisconsin Medical School, Madison, Wisconsin 53706
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ABSTRACT |
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Telokin is a 17-kDa protein with an amino acid
sequence that is identical to the COOH terminus of the 130-kDa myosin
light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene.
In the current study, we show by in situ mRNA hybridization that
telokin mRNA is restricted to the smooth muscle cell layers within
adult smooth muscle tissues. In situ mRNA analysis of mouse embryos
also revealed that telokin expression is restricted to smooth muscle
tissues during embryonic development. Telokin mRNA expression was first
detected in mouse gut at embryonic day 11.5; no telokin
expression was detected in embryonic cardiac or skeletal muscle.
Expression of telokin was also found to be regulated during postnatal
development of the male and female reproductive tracts. In both uterus
and vas deferens, telokin protein expression greatly increased between
days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the
expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and -actin.
in situ hybridization; reproductive tract development; myosin light chain kinase; embryos
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INTRODUCTION |
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SMOOTH MUSCLE CELLS
ARISE from diverse populations of precursor cells during
embryonic development, and the mechanisms that specify the smooth
muscle cell phenotype in each of these populations of cells are largely
unknown. All differentiated smooth muscle is characterized by the
presence of unique isoforms of contractile proteins such as smooth
muscle - and
-actin, myosin heavy chain, caldesmon, SM22
,
telokin, and calponin that are not expressed in other tissue types.
Analysis of the spatial and temporal patterns of expression of several
smooth muscle proteins has revealed distinct patterns of expression of
these proteins in different smooth muscle tissues (10, 14,
17-19, 24). The functional significance of these unique
patterns of expression is largely unknown. This is partly because the
physiological functions of proteins such as telokin, SM22
, and
calponin are not well established and partly because the unique
functions of contractile protein isoforms such as the smooth muscle
- and
-actins are not well understood. In our efforts to
understand the transcriptional regulation and function of telokin, we
previously demonstrated that its expression is restricted to smooth
muscle tissues, where it is expressed at high levels in visceral smooth
muscle tissues and lower levels in vascular tissues (6,
10).
Although its physiological function is unclear, telokin has been shown to bind to unphosphorylated myosin filaments and to stimulate myosin mini-filament assembly in vitro. Consequently, it has been proposed that telokin may play an important role in maintaining the stability of unphosphorylated myosin filaments in vivo (11, 27). Recently, it has been suggested that telokin induces relaxation of permeabilized ileum smooth muscle strips through activation of myosin light chain phosphatase. Phosphorylation of telokin by cAMP- or cGMP-dependent protein kinases augmented its relaxant effects (31). These studies suggest that telokin may play an important role in regulating smooth muscle contractility and that regulation of telokin expression levels may be an important mechanism for long-term modulation of contractility.
The amino acid sequence of telokin is identical to the COOH terminus of the 130-kDa "smooth muscle" myosin light chain kinase (MLCK) and the 220-kDa "nonmuscle" long form of MLCK. Telokin is not a proteolytic fragment derived from the MLCK but is the translation product of a distinct 2.6-kb mRNA (6). We have previously shown that telokin mRNA is transcribed from a second promoter located within an intron that interrupts the exons encoding the calmodulin binding domain of the MLCKs (10). Unlike the 130-kDa MLCK that has been detected in all adult tissues examined thus far, telokin protein and mRNA expression is restricted to smooth muscle tissues and cultured smooth muscle cells (6, 7, 10). Several studies have suggested that telokin is also expressed in embryonic chicken heart and in tissue samples from some, but not all, human hearts. In contrast, we have not observed detectable levels of telokin expression in adult mouse, rat, or rabbit heart (1, 5, 6, 10, 32). Because several smooth muscle contractile proteins are expressed transiently during cardiac development and many cardiac pathologies result in a reexpression of a fetal gene program (26), this raises the possibility that telokin may also be transiently expressed during cardiac development. In the current study, we have used mRNA in situ hybridization to determine the cellular localization of telokin within adult smooth muscle tissues and during mouse embryonic development. These studies reveal that telokin expression is restricted to smooth muscle cells during mouse development. In addition, induction of telokin during the postnatal differentiation of the reproductive tract paralleled the induction of other smooth muscle-specific proteins.
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MATERIALS AND METHODS |
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Cloning mouse telokin cDNA.
A 1,845-bp fragment corresponding to nucleotides 1985-3830 of the
mouse 130-kDa MLCK (9) was used to screen a mouse
7-day-old embryo/uterine cDNA library (Clontech, Palo Alto,
CA). Lambda DNA was prepared from the positive plaques using
lambdasorb (Promega, Madison, WI), and inserts were digested with
appropriate restriction endonucleases and subcloned into pGEM for
sequencing. A rapid amplification of cDNA ends-polymerase chain
reaction procedure [5'-Amplifinder rapid amplification of cDNA ends
(RACE) kit; Clontech, Palo Alto, CA] was used to extend the 5' end of
the telokin cDNA. The 3' primer used for this analysis was
complementary to nucleotides 215-233 of the cDNA in Fig.
1. Amplified cDNAs were digested with EcoR I-Sal I and ligated into pGEM3Z.
Subcloned cDNA were sequenced using T7 and SP6 sequencing primers.
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Western and Northern blotting.
Western immunoblots and Northern blots were performed as described
previously (6). The following antibodies were used for Western blotting. Anti-peptide polyclonal antibodies specific for
myosin heavy chains SM1, SM2, NMHCA, and NMHCB were generated using
peptides derived from the unique COOH termini of each molecule as
described previously (8). A monoclonal antibody to the
gizzard MLCK (Sigma clone K36) was used to detect the 130-kDa and
220-kDa MLCKs. Telokin was detected using a polyclonal antibody to
telokin described previously (6), and smooth muscle
-actin was detected using a specific monoclonal antibody (Sigma,
clone 1A4).
In situ hybridization.
Adult mouse tissues were collected, quick frozen in isopentane at
20°, mounted in tissue-freezing media (TBS, Durham, NC), and stored
at
70°. mRNA in situ hybridization was performed on 10-µm
cryosections as described previously (16, 22). Mouse embryos were collected from timed pregnant mice, fixed in 4%
paraformaldehyde at 4° overnight, embedded in paraffin, sectioned,
and processed for in situ hybridization as described above. The telokin
probe used was identical to the riboprobe used for Northern blot
analysis, except that 35S nucleotides were used for
labeling. An antisense probe was generated using T7 RNA polymerase;
sense probes were generated using SP6 RNA polymerase. Hybridization was
carried out at 50° for 16-18 h. Final wash conditions were 0.1×
SSC (15 mM NaCl, 1.5 mM Na-citrate, pH 7.0) at 37°.
Immunostaining of tissue sections.
Tissues were collected from adult mice or embryos, quick frozen in
isopentane at 20°, mounted in tissue-freezing media (TBS), and
frozen at
70°. Ten-micrometer cryosections were cut, fixed in 3.7%
formaldehyde in PBS for 5 min, permeabilized in 0.2% Triton X-100 for
5 min, and then incubated with the appropriate antibody for 2-6 h
at 37°. Following extensive washing in 50 mM Tris, pH 7.6, 150 mM
NaCl sections were incubated with fluorescein conjugated to donkey
anti-rabbit IgG for 1 h.
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RESULTS |
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Cloning of mouse telokin. A mouse telokin cDNA was identified by using a probe derived from the mouse smooth muscle MLCK to screen a 7-day-old mouse embryo cDNA library (9). Twenty-nine overlapping lambda clones were isolated and sequenced. A 2.5-kb cDNA that overlapped the COOH-terminal coding region of the previously described 130-kDa MLCK (amino acids 878-1,051) and had 80 bp of unique 5'-untranslated sequence was identified as a telokin cDNA (6). The sequence of a 1.5-kb fragment that includes the entire coding region and 5'-untranslated region is shown in Fig. 1. The remainder of the sequence of the 3'-untranslated region is not shown but has been submitted to Genbank (accession no. AF314149). 5' RACE-PCR was used to identify the 5' end of the mouse telokin mRNA in mouse uterine tissue. Sequencing 17 of these 5' cDNA clones revealed that the 5' cDNAs were clustered in 3 groups. The longest three cDNAs extended to the end of the sequence shown in Fig. 1. The other cDNAs clustered between +35 to +44 and +53 to +78.
A probe derived from the 5'-untranslated region of mouse telokin
cDNA specifically interacts with a single 2.6-kb mRNA.
A fragment of the telokin cDNA extending from nucleotides 53 to 233 was
used as a probe for Northern blot analysis of telokin expression in
adult mouse tissues (Fig. 2). Of the nucleotides included in this
fragment, only 23 nucleotides were also present in the mouse 130-kDa
and 220-kDa MLCK cDNAs (9). The probe hybridized to a
single mRNA of 2.6-kb that was expressed at high levels in colon,
ileum, stomach, uterus, and bladder. Low levels of expression were also
observed in lung, spleen, and aorta. No cross-hybridization was
observed to either the 5.6-kb mRNA encoding the 130-kDa MLCK or the
7-kb mRNA encoding the 220-kDa MLCK (9). These data
demonstrate that a probe derived from the unique 5'-untranslated region
of the mouse telokin cDNA hybridizes specifically to telokin mRNA.
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Telokin mRNA is restricted to smooth muscle cells in adult smooth
muscle tissues.
The telokin-specific probe described above was used to localize telokin
expression in adult mouse tissues by mRNA in situ hybridization. Serial
sections were reacted with antisense and sense riboprobes to telokin or
antibodies to smooth muscle myosin heavy chain 2 or smooth muscle
-actin, or stained with hematoxylin and eosin (Figs.
3 and
4). In each of the tissues
examined, including uterus, vas deferens, bladder, colon, kidney,
ureter, ovary, and trachea, telokin mRNA expression was restricted to
the smooth muscle cell layers. The identity of the smooth muscle cell
layers was determined by positive staining for smooth muscle myosin, smooth muscle
-actin, or from morphological examination of
hematoxylin- and eosin-stained sections. Although not characterized in
detail, the telokin and smooth muscle
-actin-positive cells in the
ovary reacted only weakly with antibodies to SM2 myosin heavy chain (data not shown). These cells likely represent the previously reported
smooth muscle cells of the theca (21, 29). Similar to
previous Western and Northern blot data, low levels of telokin expression were also detected in vascular smooth muscle (Fig. 4). On
all sections, sense probes produced a very low background signal (Figs.
3 and 4).
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Telokin mRNA is restricted to smooth muscle tissues during mouse
development.
To examine the developmental pattern of telokin expression, sections
from staged mouse embryos (E9.5-E16.5) were analyzed by mRNA in
situ hybridization using a telokin-specific riboprobe. This analysis
revealed that telokin mRNA was first detectable at low levels in the
gut of day 11.5 embryos (Fig.
5A). No expression was
detected in E9.5 or E10.5 embryos (data not shown). Expression in the
gut increased during embryonic development, attaining high levels by
E15.5 (Fig. 5, C and F). Telokin mRNA expression
was also seen in the bronchi of the lung from E13.5 (Fig. 5,
D and E). By embryonic day 15.5,
telokin mRNA was also expressed at high levels in the bladder,
ureter, urethra, and rectum (Fig. 5F). Throughout
development, telokin expression was restricted to the muscular layers
of the gut, bladder, and bronchi (Fig. 5, D-F). No
expression was detected in embryonic cardiac or skeletal muscle at any
stage examined. Similarly, no telokin expression was detected in the
aorta, although expression could be detected in the umbilical artery
(Fig. 5F). On all sections, sense probes produced very low
background signals (Fig. 5B, data not shown.)
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Telokin expression is induced during postnatal development of the
reproductive tract.
Results (Fig. 3) show that telokin mRNA was restricted to the
myometrium of the uterus and the smooth muscle wall of the vas deferens
in adult mice. Unlike most other smooth muscle tissues, the smooth
muscle of the reproductive tract differentiates during postnatal
development (2). To compare the induction of telokin with
that of other markers of smooth muscle differentiation during postnatal
development of the reproductive tract, Western blot analysis was
performed (Fig. 6). Results from this
analysis of uterine extracts revealed that telokin was first detectable
at low levels by neonatal day 7. Between days 7 and 14, telokin expression increased dramatically to reach
the relatively high levels that were sustained throughout the remainder
of neonatal development. The pattern of telokin expression in the
uterus paralleled that of smooth muscle myosin heavy chain isoforms,
SM1 and SM2, which first became detectable at day 7 and
increased to adult levels by day 14. Expression of smooth
muscle -actin was first detectable in N1 uteri, and its expression
continued to increase until day 14, when it reaches levels
found in adult tissue. In contrast, the 130-kDa MLCK was expressed at
high levels throughout the postnatal development of the uterus.
Expression of the 220-kDa long form of MLCK was below the levels of
detection throughout all stages of uterine development. In contrast to
smooth muscle myosin, expression of the nonmuscle myosin heavy chain B
(NMHCB) appeared to decline between days 7 and 14 of uterine development and continued to be expressed at relatively low
levels during the remainder of neonatal development. Expression levels
of nonmuscle myosin heavy chain A (NMHCA) appeared to be slightly
variable, with a peak of expression occurring between days 7 and 14, followed by a small decline to lower levels of
expression that were maintained throughout the remainder of neonatal
development (Fig. 6).
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DISCUSSION |
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We have previously shown that telokin is exclusively expressed in adult smooth muscle tissues and in smooth muscle-derived cell lines (6, 10). The current data extend these findings and demonstrate that telokin is expressed in the smooth muscle cells of smooth muscle tissues in adult mice. In addition, in situ hybridization analysis of mouse embryos establishes that telokin expression is restricted to smooth muscle tissues during development.
Telokin was first detectable in the developing gut at embryonic
day 11.5, before the expression of most other smooth muscle markers. For example, smooth muscle myosin heavy chain, calponin, SM22, and smooth muscle
-actin were not detected until embryonic days 12.5, 13.5, 13.5, and
15.5, respectively (14, 17-19, 23, 24).
Only smooth muscle
-actin, which was detectable at embryonic day 9 in hindgut, was expressed before telokin during
embryonic visceral smooth muscle development. Unlike many other smooth
muscle proteins, telokin was not detected in adult or embryonic
skeletal muscle or in skeletal muscle-derived cell lines (Figs. 2 and 4 and Refs. 6 and 10). Previous reports using Northern
blotting and RT-PCR analysis of chicken and human samples (1, 5, 27) have suggested that telokin is expressed in embryonic and adult heart. However, our current results from in situ hybridization analysis of mouse tissues (Figs. 2 and 5), together with previous Western blot analysis (10) and RNase protection analysis
of rabbit RNA (6), suggest that in mouse and rabbit,
telokin is not expressed at detectable levels in cardiac muscle.
Although these analyses do not rule out the possibility that telokin is expressed at low levels in these tissues, the relative levels of
expression are clearly much lower than those reported in chicken and
human (1, 5, 27). Together, these results suggest that the
differences between the pattern of telokin expression in chicken and
mouse or rabbit are likely to be the result of species differences in
gene regulatory elements. This suggestion is supported by the high
degree of sequence divergence observed between chicken and rabbit
telokin promoters (28). However, the high homology between
the rabbit and human telokin promoters would suggest that it is
unlikely that differences in gene regulatory elements can explain the
differences in telokin expression observed between these two species
(28, 30). However, it is possible that the inconsistency
in telokin expression in human heart may reflect variability in the
amount of vascular smooth muscle contamination. Alternatively specific
pathological conditions may result in upregulation of telokin
expression in human heart. Additional human studies will be required to
resolve this issue.
Our data demonstrate that telokin is an exclusive marker of smooth
muscle lineages in mouse. In contrast to smooth muscle myosin heavy
chain, telokin is not detectable in embryonic dorsal aorta (Fig. 5),
although it can be detected at low levels in adult aorta (Fig. 2),
perhaps suggesting that telokin expression in aorta may be increased
later in development. Telokin expression was observed at high levels in
the umbilical artery of embryonic mice and at low but detectable levels
in vessels of the kidney (Figs. 4 and 5) and mesentery of adult animals
(data not shown). The pattern of telokin expression most closely
parallels that of the smooth muscle -actin, which is also expressed
at high levels in visceral compared with vascular smooth muscle and is also not expressed in embryonic cardiac or skeletal muscle (17, 25). Telokin, however, in contrast to smooth muscle
-actin, is not expressed at detectable levels in testes (data not shown; Ref. 12). These data show that although telokin,
smooth muscle
-actin, and smooth muscle myosin heavy chain are
characteristic markers of smooth muscle cells, they exhibit
differential levels of expression in distinct smooth muscle tissues.
This suggests that it is likely that distinct as well as overlapping
mechanisms control the expression of genes in vascular compared with
visceral smooth muscle. This proposal is further supported by data from transgenic mice in which fragments of the SM22
promoter have been
used to drive
-galactosidase expression (13,
15). Analysis of these animals revealed that the promoter
fragments analyzed were sufficient to direct expression to vascular but
not visceral smooth muscle, although the endogenous SM22
was
expressed in both tissues. This suggests that additional regulatory
elements are required for expression in visceral smooth muscle compared with vascular smooth muscle. The expression of telokin in distinct vascular beds, together with the expression of SM22
transgenes in
arterial but not venous vessels, further suggests that distinct regulatory mechanisms may also operate in different regions within the
vascular system. The heterogeneity of regulatory mechanisms likely
reflects the diverse embryological origins of smooth muscle cells in
these different tissues.
To complete the analysis of the developmental expression of telokin, we
also examined telokin expression during postnatal development of the
reproductive tract. Unlike other smooth muscle tissues, the smooth
muscle of the male and female reproductive tracts differentiates during
postnatal development. However, a comprehensive analysis of the pattern
of smooth muscle protein expression during this postnatal period has
not been previously conducted. In the current study, we characterized
the expression of telokin, MLCK, -actin, and myosin heavy chain
isoforms during postnatal development of uterus and vas deferens by
Western blotting. In both tissues, there was a relative increase in
expression of smooth muscle myosins, actin, and telokin up to maximal
levels by neonatal day 14 (Figs. 6 and 7). Paralleling
embryonic vasculature, smooth muscle
-actin expression preceded
expression of other smooth muscle markers in the developing uterus. In
the vas deferens, however, the relative increase in the expression
levels of SM1 and SM2 paralleled the increase in smooth muscle
-actin expression. This suggests that there may be subtle
differences in the regulation of smooth muscle differentiation in the
female and male reproductive tracts. In both uterus and vas deferens,
no further increases in expression of smooth muscle markers were
observed between neonatal days 14 and 25. In
contrast to the expression of smooth muscle-specific proteins,
expression of NMHCA and NMHCB declined during postnatal development,
and expression of the ubiquitous 130-kDa MLCK remained relatively
constant. During development in chicken, expression of the 130-kDa MLCK
was preceded by the expression of a 206-kDa embryonic isoform
(analogous to the mouse 220-kDa MLCK) (5). In the current
study, expression of the 220-kDa MLCK was below the levels of detection
in both uterus and vas deferens throughout the postnatal developmental
period analyzed. The 220-kDa MLCK was thus not expressed at high levels
in the reproductive tract of newborn mice, at a time when other markers
of embryonic or undifferentiated smooth muscle were present in
abundance. These data suggest that either expression of the 220-kDa
MLCK was downregulated before birth in the mammalian reproductive tract
or that it was not expressed at high levels in these tissues.
In general, the changes in myosin heavy chain expression in uterus and vas deferens are consistent with those reported previously in mouse uterus (4). The nonmuscle type pattern of protein expression in uteri and vas deferens from newborn mice is consistent with the morphological identification of these organs as epithelial tubes surrounded by undifferentiated stromal cells (3). The induction of smooth muscle-specific proteins at day 7 correlates with previous morphological identification of smooth muscle differentiation in these tissues (2). The induction of smooth muscle differentiation also correlates with the specialization of uterine epithelia and the downregulation of wnt4 and wnt5a in the differentiating myometrium (20). Extensive studies have shown that epithelial-stromal interactions are important for the development of the reproductive tract, and recent studies have suggested that wnt signaling may play a role in this process. The postnatal development of the reproductive tract thus provides a useful model system for studying the role of wnt signaling in the induction of smooth muscle differentiation.
In summary, our results show that telokin expression was restricted to smooth muscle cells during murine development. These findings show that telokin is a specific marker of smooth muscle differentiation.
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ACKNOWLEDGEMENTS |
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We thank Gina Simon and Bruce K. Micales for expert technical assistance.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-58571 (to B. P. Herring) and HL-54118 (to P. J. Gallagher).
G. E. Lyons is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: B. P. Herring, Dept. of Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202 (E-mail: pherring{at}iupui.edu).
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.
Received 26 May 2000; accepted in final form 8 August 2000.
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