Significance Reactivating sleep-like quiescent cells in the body (most notably stem cells) to divide is fundamental to tissue homeostasis and regeneration. Like sleep having shallow and deep stages, quiescence exhibits graded depths with different speeds and rates responding to stimulation signals. Understanding how quiescence depth is regulated is fundamental to our understanding of tissue repair and regeneration. Here we show that quiescence depth is regulated like a “dimmer switch” by lysosomes (cell organelles that break down and recycle biomolecules), through the lysosomal function of reducing oxidative stress. We developed a gene signature to predict quiescence depth and show that quiescence deepening likely represents a transition path from cell division to irreversibly arrested senescence, related to the aging process.

Abstract The reactivation of quiescent cells to proliferate is fundamental to tissue repair and homeostasis in the body. Often referred to as the G0 state, quiescence is, however, not a uniform state but with graded depth. Shallow quiescent cells exhibit a higher tendency to revert to proliferation than deep quiescent cells, while deep quiescent cells are still fully reversible under physiological conditions, distinct from senescent cells. Cellular mechanisms underlying the control of quiescence depth and the connection between quiescence and senescence are poorly characterized, representing a missing link in our understanding of tissue homeostasis and regeneration. Here we measured transcriptome changes as rat embryonic fibroblasts moved from shallow to deep quiescence over time in the absence of growth signals. We found that lysosomal gene expression was significantly up-regulated in deep quiescence, and partially compensated for gradually reduced autophagy flux. Reducing lysosomal function drove cells progressively deeper into quiescence and eventually into a senescence-like irreversibly arrested state; increasing lysosomal function, by lowering oxidative stress, progressively pushed cells into shallower quiescence. That is, lysosomal function modulates graded quiescence depth between proliferation and senescence as a dimmer switch. Finally, we found that a gene-expression signature developed by comparing deep and shallow quiescence in fibroblasts can correctly classify a wide array of senescent and aging cell types in vitro and in vivo, suggesting that while quiescence is generally considered to protect cells from irreversible arrest of senescence, quiescence deepening likely represents a common transition path from cell proliferation to senescence, related to aging.

Cell proliferation in multicellular organisms is tightly regulated, and the vast majority of cells in the body stay dormant and out of the cell cycle at any given moment. The dormant state, when reversible upon growth signals, is referred to as cellular quiescence. Quiescence protects cells against stress and irreversible arrest, such as senescence; it is fundamental to many physiological phenomena, such as stem cell homeostasis and tissue repair (1⇓⇓–4). Consequently, dysregulation of cellular quiescence can lead to a range of hyper- and hypoproliferative diseases, including cancer and aging (5⇓–7).

It becomes increasingly recognized that quiescence is a heterogeneous state with graded depth. For example, following injury, muscle and neural stem cells at noninjury sites in the body move adaptively into shallower quiescence (alert or primed quiescent phase), positioning cells to more quickly respond to injury and reenter the cell cycle when needed (8, 9). In other cases, cells move into deep quiescence and require stronger growth stimulation and longer times to reenter the cell cycle, as seen in hepatocytes upon partial hepatectomy in older rats than in younger ones (10, 11). Similarly, the quiescent state deepens in cells that are cultured longer under quiescence-inducing signals, such as contact inhibition (12, 13) and serum starvation (14). Deep quiescent cells can still revert to proliferation under physiological conditions, appearing distinct from irreversibly arrested senescent cells (SI Appendix, Fig. S1A). A graded quiescence depth indicates graded potentials for tissue repair and regeneration; yet, how quiescence depth is regulated in the cell and the connection between deep quiescence and senescence are not well understood.

In this study, we investigate what regulates quiescence depth in rat embryonic fibroblast (REF) cells. We identified sequential transcriptome changes as cells move progressively deeper into quiescence under longer-term serum starvation. In particular, we found that lysosomal gene expression and lysosomal mass (indicated by the intensity of lysosomal staining and the number of lysosomes per cell) continuously increase as quiescence deepens; however, autophagy flux decreases. Lysosomes are hydrolytic enzyme-filled organelles in the cell that break down many types of biomolecules; lysosomal function, primarily through processes of autophagy and endocytosis, has been shown to prevent irreversible cellular states, such as senescence, terminal differentiation, and apoptosis (15⇓⇓–18). Here we found that the increased lysosomal mass in deep quiescent cells is, in part, due to decreased autophagy flux, which is partially but not fully compensated by increased lysosomal gene expression and lysosomal biogenesis. We show that lysosomal function, like a dimmer switch, continuously regulates quiescence depth and thus the proliferative potential of quiescent cells by reducing the accumulation of reactive oxygen species (ROS). We found that a set of “senescence core signature” genes (19) show similar expression patterns in deep quiescence as in senescence, and that a gene-expression signature we developed to indicate cellular quiescence depth by comparing deep and shallow quiescent REF cells is able to correctly classify senescent and aging cells in a wide array of cell lines in vitro (20, 21) and tissues in vivo (22, 23), suggesting that deep quiescence may serve as a common transition path from cell proliferation to senescence, related to aging.

Materials and Methods Detailed descriptions of RNA-seq and downstream data analysis and modeling, modulation of lysosomal–autophagic function and lysosome biogenesis, assays for quiescence-depth, lysosomal mass and proteolytic activity, autophagy flux, mitochondrial ROS, β-galactosidase activity, cell size, and cytotoxicity are provided in SI Appendix. Cell Culture and Quiescence Induction. REFs used in this study are from a single-cell clone derived from REF52 cells and contain a stably integrated human E2F1 promoter-driven destabilized EGFP (E2f-GFP) reporter, as previously described [i.e., REF/E23 cells (25, 82)] unless otherwise noted. Cells were passaged every 2 to 3 d and maintained at subconfluency in growth medium: DMEM supplemented with 10% bovine growth serum (BGS; GE Healthcare, SH30541). To induce quiescence, cells were plated at ∼50% confluence in growth medium for a day, washed twice with DMEM, and cultured in serum-starvation medium (DMEM containing 0.02% BGS) for the indicated duration (≥2 d). Quiescence-Depth Assay. To assess quiescence depth, cells were switched from serum-starvation medium to serum-stimulation medium (DMEM containing BGS at indicated concentrations) and harvested at indicated time points by trypsinization. The cell fraction that reentered the cell cycle was quantified by assessing the profile of EdU incorporation (and the profiles of E2f-GFP reporter and PI DNA staining when indicated). Quiescence depth is determined by the serum threshold required to activate cells to reenter the cell cycle. At a given serum concentration, a smaller percentage of deeper quiescent cells are able to reenter the cell cycle by a given time than of shallower quiescent cells. See text for details. Data Availability. The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE124109).

Acknowledgments We thank Johnny Fares for valuable discussions and suggestions on lysosomal function and assays; Andrew Peak and Andrew Capaldi for critical reading and comments on the manuscript; and Haoxing Xu for providing Mcoln1 inhibitors. This work was supported by National Science Foundation Grants DMS-1463137 (to G.Y.), DMS-1418172 (to G.Y. and H.H.Z.), CCF-1740858 (to H.H.Z.), and DMS-1462049 (to J.X.); National Institutes of Health Grant R01DK119232 (to J.X.); Chinese National Science and Technology Major Project 2018ZX10302205 (to F.B.); and Guangdong Province Key Research and Development Program 2019B020226002 (to F.B.).

Footnotes Author contributions: K.F., R.L., J.X., F.B., and G.Y. designed research; K.F., R.L., H.C., and K.D.C. performed research; K.F., R.L., H.C., K.D.C., H.H.Z., J.X., F.B., and G.Y. analyzed data; and K.F., F.B., and G.Y. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE124109).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1915905116/-/DCSupplemental.