Research reportDistribution of the transcription factor Signal Transducer and Activator of Transcription 3 in the rat central nervous system and dorsal root ganglia
Introduction
The family of Signal Transducer and Activator of Transcription (STAT) proteins is a group of transcription factors that are activated (i.e., phosphorylated at a tyrosine residue) following binding of certain cytokines to their respective cell membrane receptors. Following such activation, the STAT proteins homo- or hetero-dimerize and translocate to the nucleus where the STAT proteins are involved in transcriptional regulation, both directly by binding DNA and indirectly by protein–protein interactions with other DNA-binding factors 4, 29.
STAT3 was first identified through its partial homology with STAT1α [41]. There are two differently spliced isoforms of STAT3, STAT3α (92 kDa) and STAT3β (83 kDa, missing the C-terminal 55 amino acids) 27, 41. Both forms have an SH2, an SH3, and an LXXLL domain, that can mediate interactions with other proteins (e.g., cell surface receptors, transcription factors such as STAT1, and steroid hormone receptors), and in addition a DNA binding domain and a trans-activating domain 4, 35, 39. However, the two isoforms differ in their pattern of activation, the stability of their phosphorylation at tyrosine residues, and their trans-activating potential [28]. STAT3 can be activated by the receptors for a number of polypeptide ligands, for example interleukin 6 (IL-6) [41], oncostatin M [17], leukemia inhibitory factor (LIF) [16], ciliary neurotrophic factor (CNTF) [25] and leptin 8, 26.
Activated STAT3 can bind to the serum inducible element in the c-fos gene promoter [41], to the jun-B promoter [6], to the preproenkephalin gene promoter [13], and, providing a negative feed-back to STAT3 activation, to the promoter of the family of STAT-induced STAT inhibitor proteins (the SOCS family) 21, 32. In sympathetic neurons, activated STAT3 has been shown to bind to cytokine response elements [33].
STAT3 mRNA has been shown to be expressed in the rat brain [40]. STAT3 protein is expressed constitutively in the rat cerebellum and may play a role in maintaining basal c-fos expression [24]. Intravenously administrated interleukin 1β and leptin have been shown to induce tyrosine phosphorylation of STAT3 in the rat paraventricular and arcuate hypothalamic nuclei, respectively 13, 36, and CNTF stimulation of chick ciliary ganglion neurons results in a similar phosphorylation event [38].
However, the basal expression and distribution of STAT3 protein in the intact rat nervous system is still unknown. Such knowledge is necessary for the understanding of where cytokines affect nervous system function and how they exert their effect at the cellular level. In the present study, the distribution of STAT3 protein has been studied using immunohistochemical techniques.
Section snippets
Animals
Six adult male Sprague–Dawley rats (B&K Universal, Stockholm, Sweden; 200–400 g) were used. They were housed two to a cage under 12-h light:12-h dark conditions with water and food available ad libitum. All the animals were killed during the dark period not longer than 1 h after they had been removed from their ordinary environment. The experiments were approved by the Animal Care and Use Committee of the University of Linköping, Sweden.
Tissue preparation
The rats received a lethal dose of sodium pentobarbital
Antibody specificity
Western blotting with antigen detection using the Santa Cruz antiserum showed strong labeling of a band at approximately 92 kDa (Fig. 1a). Antigen detection using the New England Biolabs antiserum resulted in moderate labeling of a band at approximately 92 kDa, but in addition there was strong labeling of a second band at a higher molecular weight (Fig. 1b).
Preincubation of the primary antiserum (Santa Cruz) with the control peptide before immunohistochemistry abolished all labeling (Fig. 2a,b).
Antibody specificity
The results of Western blotting demonstrated that two antisera directed at different epitopes of STAT3 each labeled a protein at a molecular weight of approximately 92 kDa, corresponding to that of STAT3. When used for immunohistochemistry, both antisera produced similar labeling patterns. These observations strongly suggest that the distribution of STAT3-LI, as seen in brain sections, reflect the distribution of STAT3 protein in the brain.
Subcellular distribution of STAT3-LI
The observations of STAT3-LI in neuronal cell bodies
Acknowledgements
This work was supported by the Swedish Medical Research Council (#7879), the Swedish Foundation for Strategic Research (H.S.), the Swedish Natural Science Council (S.P.S.S.), and the Swedish Brain Foundation (O.H.). We thank Dr. Anders Blomqvist for comments on the manuscript, and Martin Hallbeck for technical support.
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