Interferon (IFN) –alpha stimulates the accumulation of Myxovirus (influenza virus) resistance 1 (Mx1) pseudo-gene transcripts including an intron-containing transcript in mouse pituitary gonadotropes

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-160
Ryota Terashima*1, Titaree Laoharatchatathanin1, Tomohiro Yonezawa1, Shiro Kurusu1, Takako Kato2, Yukio Kato2 and Mitsumori Kawaminami1
1Kitasato Univ, Aomori, Japan, 2Meiji Univ, Kanagawa, Japan
Myxovirus (influenza virus) resistance 1 (Mx1) is a major target gene of IFN-alpha, but is usually a pseudo-gene in laboratory mice (M21039.1, M21038.1). We investigated, in the present study, the effect of IFN-alpha on pituitary gonadotropes with special reference to a putative role of Mx1 pseudo-gene. The expression of 5’ intact portion of Mx1 mRNA showed that the transcription of the pseudo-gene was stimulated by IFN-alpha in LβT2 mouse gonadotrope cells. IFN-alpha suppressed the growth of LβT2 cells in a dose and time dependent manner. IFN-alpha was also shown to augment LH release and LHb mRNA expression by 6 hour stimulation with GnRH of LβT2 cells. The inhibitory effect of IFN-alpha on cell growth was suppressed by a small interfering RNA targeting Mx1. 3’ RACE analysis demonstrated at least five Mx1 related transcripts in LβT2 cDNAs. Four of them were all truncated mRNA sequences of Mx1, but one of transcripts consisted of exon 1, 2, 3 and a part of intron 3 that makes a new open-reading frame with a stop codon and polyadenylation site. This intron containing-transcript was seen in the pituitary glands of both MSM/Ms and C57BL6 mice. MSM/Ms is a wild derived inbred mouse with intact Mx1 gene and C57BL6 is a typical laboratory mouse with the pseudo-gene. Hence, the intron containing-transcript is suggested to be a normal variation of Mx1 gene transcription. Increase of this intron containing-transcript was most apparent among five transcripts after IFN-alpha stimulation of LβT2. Immunohistochemistry showed that the intra-nuclear localization of Mx1 only in the pituitary gland of MSM/Ms but rare in C57BL6. This suggests that the pseudo-gene has lost a sequence necessary for nuclear translocation and the inhibition of cell growth is performed in cytosol fraction. Finally, we made expression vectors with pcDNA3.1 for the Mx1 pseudo-gene and the intron containing-transcript. Both were shown to suppress LβT2 cell growth. These data clearly demonstrate that gene products of so-called pseudo-Mx1 are functional in the pituitary gonadotropes and suggest that a splicing variation would be an important mechanism for the action of IFN-alpha. As we confirmed that IFN-alpha was synthesized in LβT2 cells, an autocrine regulation by IFN-alpha and Mx1 transcripts is suggested.

Nothing to Disclose: RT, TL, TY, SK, TK, YK, MK

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