Telokin expression is restricted to smooth muscle tissues during mouse development

Abstract

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.

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.

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.

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.

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 α-actin (α-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.

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, non-specific signal from red blood cells; NT, neural tube; TEL, telencephalon; FV, fourth ventricle; VENT, cardiac ventricle; BLAD, bladder; UA, umbilical artery; UR, urethra.

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 α-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/α-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.

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.