Fibroblast PER2 circadian rhythmicity depends on cell density

Abstract

Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high- and low-density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low-density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these 2 possibilities, we mixed PER2::LUC wild-type (WT) cells with nonluminescent, nonrhythmic Bmal1-/- cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high-density cultures. We also mixed PER2::LUC WT cells with nonluminescent, long period Cry2-/- cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or nonrhythmic Bmal1-/- cells. In previous work, we found that low K⁺ suppresses fibroblast rhythmicity, and we and others have found that either low K⁺ or low Ca²⁺ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low-density fibroblasts with high K⁺ (21 mM), high Ca²⁺ (3.6 mM), or conditioned medium. Conditioned medium from high-density fibroblast cultures rescued rhythmicity of low-density cultures, whereas high K⁺ or Ca²⁺ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity, but that these signals do not have to be rhythmic, and that rhythmic signals from other cells do not affect the intrinsic periods of fibroblasts.

Keywords: PER2; circadian rhythms; density; fibroblasts; imaging; luciferase.

Figure 1

Fibroblasts lose PER2::LUC rhythmicity in low density culture. Bright field images (above) and representative PER2::LUC bioluminescence intensity patterns (below) of fibroblasts in cultures with cell densities of 27 cells/mm² (A), 46 cells/mm² (B), and 150 cells/mm² (C). Imaging began immediately following a change to fresh explant medium (day 0).

Figure 2

Circadian rhythm parameters in low and high density cultures: percentage of rhythmic cells for cultures of various cell densities (A), percentage of rhythmic cells (B), brightness of cells (C), and (for rhythmic cells) period (D) and amplitude (E), in cultures of low density (< 27 cells/mm²) and high density (> 46 cells/mm²). Columns show average values ± SEM, and white numerals inside columns indicate numbers of cultures (B) or numbers of cells (C–E). ns, not significant; **p < 0.01 (Mann-Whitney U-test).

Figure 3

PER2::LUC fibroblasts were mixed with a 20- to 100-fold excess of non-luminescent fibroblasts from WT, Bmal1−/−, or Cry2−/− mice. Representative bright field (A) and bioluminescence (B) images of mixed fibroblast cultures are shown. Percentage of rhythmic PER2::LUC cells (C), brightness of cells (D), and (for rhythmic cells) period (E) and amplitude (F), in mixed cultures with WT, Bmal1−/−, or Cry2−/− fibroblasts. Columns show average values ± SEM, and numerals within columns indicate number of cultures (C) or number of cells (D-F). ns, not significant.

Figure 4

Circadian rhythm parameters in low and high density cultures without supplement, and in low density cultures with high K⁺ (final 21 mM), high Ca²⁺ (final 3.6 mM), or 50% conditioned medium: percentage of rhythmic cells (A), brightness of cells (B), and (for rhythmic cells) period (C) and amplitude (D). Columns show average values ± SEM, and numerals within columns indicate number of cultures (A) or number of cells (B-D). Low, low density culture; K⁺, high K⁺, Ca²⁺, high Ca²⁺; CM, conditioned medium; ctl, control; ns, not significant; *p < 0.05; **p < 0.01 (ANOVA followed by Dunnett’s test). Significance levels for comparisons to low density cultures are shown in black, and comparisons to high density cultures in gray. At right (E), representative PER2::LUC bioluminescence patterns of cultured fibroblasts at low density with regular medium (top), at low density with conditioned medium (middle), and at high density (bottom).