Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways William J. Hurst,* David Earnest,† and Martha U. Gillette* , ‡ , § ,1 *Department of Cell and Structural Biology, ‡Department of Molecular and Integrative Physiology, and §Neuroscience Program, University of Illinois at Urbana–Champaign, 601 South Goodwin Avenue, Urbana, Illinois 61801; and †Department of Human Anatomy, Texas A&M University Health Science Center, College of Medicine, College Station, Texas 77843-1114 Received January 10, 2002 We undertook an extensive antigenic characteriza- tion of the SCN 2.2 cell line in order to further evaluate whether the line expresses components of circadian regulatory pathways common to the hypothalamic su- prachiasmatic nucleus (SCN), the central circadian clock in mammals. We found that differentiated SCN 2.2 cultures expressed a broad range of putative clock genes, as well as components of daytime, nighttime, and crepuscular circadian regulatory pathways found within the SCN in vivo. The line also exhibits several antigens that are highly expressed in a circadian pat- tern and/or differentially localized in the SCN relative to other hypothalamic regions. Expression of a broad complement of circadian regulatory proteins and pu- tative clock genes further support growing evidence in recent reports that the SCN 2.2 cell line is an appro- priate model for investigating the regulation of cen- tral mammalian pacemaker. © 2002 Elsevier Science (USA) Key Words: period; cryptochrome; casein kinase; PACAP; PKA; PKC; PKG; NOS; I1; VGF. The central biological pacemaker in mammals lies in the hypothalamic suprachiasmatic nucleus (SCN). The SCN drives circadian behavior, expresses rhythmic gene expression and integrates external stimuli in or- der to synchronize molecular timekeeping mechanisms with changing environmental conditions. Receptivity to phase shifting stimuli is gated by the circadian state of the central pacemaker. This gating behavior is most clearly demonstrated by the sensitivity of the SCN to light during the night phase, but not during the day phase. All photic, social and hormonal influences on the circadian clock are integrated at the cellular level through multiple signaling pathways within the SCN. The complexity of interacting signaling pathways involved in regulation of the SCN has encouraged re- searchers to seek cell culture systems to complement in vivo analysis of the mammalian circadian clock. Whether a cell line exhibits central or peripheral cir- cadian clock characteristics complicates selection of an appropriate model system. To provide useful applica- tions for central mammalian pacemaker research, cell line models must exhibit persistent self-sustained os- cillations in circadian gene products, restore animal circadian rhythms in SCN-lesioned hosts and exhibit time-dependent responses to stimuli through regula- tory pathways that characterize SCN function. Rhyth- mic gene expression has been demonstrated in NIH/ 3T3 fibroblasts, Rat-1 fibroblasts and spontaneously immortalized embryonic mouse fibroblasts after syn- chronizing signals (1–5). However, there is no pub- lished evidence that these lines express spontaneous, self-sustained rhythms or rescue rhythms in SCN- lesioned animals (5, 6). In contrast, another cell line derived from fetal rat SCN, the SCN 2.2 cell line, exhibits endogenous spontaneous circadian rhythmic- ity in vitro (5, 7). We have focused on the SCN 2.2 line to further assess its usefulness as a model system for studying the mammalian central circadian pacemaker. The SCN 2.2 cell line is a pluripotent and immortal- ized line of SCN progenitor cells from fetal rat hypo- thalamic tissue that exhibits both neuronal and glial morphologies in culture (5, 7). Neuronal cell types ex- press several peptide neurotransmitters found in the SCN. These include vasoactive peptide (VIP), arginine- vasopressin (AVP), and somatostatin (SMT). Addition- ally, the line lacks oxytocin- (OXY) and corticotropin- releasing factor- (CRF) staining, which are found in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus but not in the nearby SCN (7). Like the SCN in situ, SCN 2.2 cultures generate robust twenty-four hour rhythms in brain-derived neurotro- phic factor (BDNF) and neurotrophin-3 (NT3) content 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 217-244-1648. E-mail: mgillett@uiuc.edu. Biochemical and Biophysical Research Communications 292, 20 –30 (2002) doi:10.1006/bbrc.2002.6589, available online at http://www.idealibrary.com on 20 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.