Nuclear localization of STAT5A modified with O-linked N-acetylglucosamine and early involution in the mammary gland of Hirosaki hairless rat.
Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.
Nanashima
Naoki
N
Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Asano
Jumpei
J
Hayakari
Makoto
M
Nakamura
Toshiya
T
Nakano
Hajime
H
Yamada
Toshiyuki
T
Shimizu
Takeshi
T
Akita
Miki
M
Fan
Yang
Y
Tsuchida
Shigeki
S
D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism; D000373:Agglutinins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D006579:Heterozygote; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism*; D010766:Phosphorylation; D011388:Prolactin / Q000097:blood; D051381:Rats; D050799:STAT5 Transcription Factor / Q000096:biosynthesis*; D012685:Sepharose / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014908:Triticum / Q000378:metabolism
D000373:Agglutinins; D018076:DNA, Complementary; D037102:Lectins; D050799:STAT5 Transcription Factor; D011388:Prolactin; D012685:Sepharose; D000117:Acetylglucosamine