Telokin expression is restricted to smooth muscle tissues during mouse development

B. Paul Herring1, Gary E. Lyons2, April M. Hoggatt1, and Patricia J. Gallagher1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -actin.

in situ hybridization; reproductive tract development; myosin light chain kinase; embryos


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha - and gamma -actin, myosin heavy chain, caldesmon, SM22alpha , 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, SM22alpha , and calponin are not well established and partly because the unique functions of contractile protein isoforms such as the smooth muscle alpha - and gamma -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Nucleotide and translated amino acid sequence of mouse telokin. The nucleotide sequence of mouse telokin shown is a composite of sequence obtained from a 1.4-kb cDNA clone fragment together with the 5' sequence obtained by rapid amplification of cDNA ends (RACE)-PCR. The nucleotide sequence 5' of the arrow is unique to telokin and not present in the mouse myosin light chain kinase (MLCK) cDNAs (9). The region complementary to the oligonucleotide used to extend the 5' end of the cDNA using 5'-RACE is underlined. The sequence of the remaining 1 kb of 3'-untranslated region is not shown.

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 alpha -actin was detected using a specific monoclonal antibody (Sigma, clone 1A4).

Northern blots were probed with a 180-base mouse telokin cRNA corresponding to nucleotides 53-233 of the mouse telokin cDNA. Final wash conditions for Northern blotting were 1.25 mM sodium phosphate, pH 7.4, 30 mM NaCl, 0.2 mM EDTA (0.1× sodium chloride-sodium phosphate-EDTA), and 0.1% SDS at 68° for 10 min. Blots were exposed for 2 days at -70°.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   A probe derived from the 5'-untranslated region of a telokin cDNA specifically hybridized to a single 2.6-kb mRNA. Northern blot analysis of total RNA isolated from the indicated mouse tissues. Top: the Northern blot was reacted with a riboprobe complementary to the 5'-untranslated region of telokin (nucleotides 53-233) and exposed to film for 12 days. This probe specifically reacted with a 2.6-kb mRNA that is expressed at high levels in colon, ileum, stomach, uterus, and bladder. Bottom: ethidium bromide staining of 18S rRNA.

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 alpha -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 alpha -actin, or from morphological examination of hematoxylin- and eosin-stained sections. Although not characterized in detail, the telokin and smooth muscle alpha -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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Fig. 3.   Telokin mRNA is localized to smooth muscle cells in adult mouse urinary and reproductive tract tissues. In situ mRNA hybridization analysis of telokin expression in mouse uterus, vas deferens, and bladder as indicated. Serial sections cut from each of the tissues were reacted with antisense (AS) and sense (S) riboprobes specific for telokin, antibodies to SM2 smooth muscle myosin heavy chain (SM2), or stained with hematoxylin and eosin (H & E). Sections reacted with riboprobes were visualized under dark-field illumination to visualize the silver grains representing telokin mRNA. Sections reacted with antibodies to smooth muscle myosin heavy chain were visualized by epifluorescence following incubation with fluorescein-conjugated second antibodies as described in MATERIALS AND METHODS. Hematoxylin- and eosin-stained sections were visualized by bright-field microscopy. All panels are same scale with the scale bar representing 100 µm.



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Fig. 4.   Telokin mRNA is localized to smooth muscle cells in adult mouse tissues. In situ mRNA hybridization analysis of telokin expression in mouse colon, ureter, ovary, kidney, and trachea as indicated. Serial sections cut from each of the tissues were reacted with antisense and sense riboprobes specific for telokin, antibodies to SM2 smooth muscle myosin heavy chain, smooth muscle alpha -actin (alpha -ACTIN), or stained with hematoxylin and eosin (H & E) as indicated and described in Fig. 3. In the kidney sections, the positions of 2 arteries (A) and the ureter (U) are indicated. The background signal around the arteries observed on sense sections of kidney results from the high refractance of connective tissue. All panels are same scale with the scale bar representing 100 µm.

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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Fig. 5.   Telokin mRNA is restricted to smooth muscle tissues during mouse development. In situ hybridization analysis of telokin expression in mouse embryos. Sections obtained from mouse embryos at E11.5 (A and B), E12.5 (C), E13.5 (D), and E15.5 (E and F) were hybridized with sense (B) or antisense (A and C-F) riboprobes that specifically react with telokin. Specimens were processed for in situ hybridization as described in MATERIALS AND METHODS and photographed under dark-field illumination. The control sense riboprobe is representative of the low background staining (B). NS, nonspecific signal from red blood cells; NT, neural tube; TEL, telencephalon; FV, fourth ventricle; VENT, cardiac ventricle; BLAD, bladder; UA, umbilical artery; UR, urethra.

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 alpha -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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Fig. 6.   Telokin expression is induced during postnatal development of the uterus. Western blot analysis of telokin and several other contractile proteins in uteri from neonatal mice of various ages from 1 to 30 days after birth (N1-N30). Samples were obtained from 3 mice at each time point (only 2 N30 samples are shown). Five micrograms of extract were analyzed in each lane. Samples for analysis of myosin heavy chain and MLCK were separated on 5% polyacrylamide gels and those for telokin and alpha -actin on 15% gels. To facilitate comparisons between blots, the third sample at N14 shown (left) is repeated in lane 1 (right). A single pair of blots was used for the analysis of each of the myosin heavy chain isoforms and MLCK. After each blot was reacted with an antibody, it was stripped and reprobed with second-step antibody to confirm that the antibody had been stripped from the blots before reacting with a subsequent antibody. The same procedure was used for the telokin/alpha -actin blots. The positions of molecular mass markers are indicated to the left of the blots. NMHCA, nonmuscle myosin heavy chain A; NMHCB, nonmuscle myosin heavy chain B.

The postnatal pattern of expression of smooth muscle contractile proteins in vas deferens was very similar to that observed in uterus (Fig. 7). The expression of telokin, SM1, SM2, and smooth muscle alpha -actin reached maximal levels between postnatal days 7 and 14. NMHCA and NMHCB were expressed at high levels in vas deferens from newborn mice. NMHCA expression began to decline between days 7 and 14, and its expression continued to decline throughout the developmental period analyzed. The decline in NMHCB expression, between days 1 and 7, occurred earlier than the decline in NMHCA expression. In contrast to the other proteins examined, the 130-kDa MLCK was maintained at similar levels throughout neonatal development. No expression of the 220-kDa MLCK was detected at any stage examined (Fig. 7).


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Fig. 7.   Telokin expression is induced during postnatal development of the vas deferens. Western blot analysis of telokin and several other contractile proteins in vas deferens from neonatal mice of various ages from 1 to 25 days after birth (N1-N25). Analysis was performed as described in Fig. 6 except that 10 µg of extract was analyzed in each lane of the 5% gels and 2 µg were analyzed on each lane of the 15% gels. The samples at N14 were loaded identically onto each of the 2 blots analyzed. The positions of molecular mass markers are indicated to the left of the blots.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, SM22alpha , and smooth muscle gamma -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 alpha -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 gamma -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 gamma -actin, is not expressed at detectable levels in testes (data not shown; Ref. 12). These data show that although telokin, smooth muscle gamma -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 SM22alpha promoter have been used to drive beta -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 SM22alpha 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 SM22alpha 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, alpha -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 alpha -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 alpha -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.


    ACKNOWLEDGEMENTS

We thank Gina Simon and Bruce K. Micales for expert technical assistance.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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