Endogenous histidyl dipeptides such as carnosine (β-alanine-L-histidine) form conjugates with lipid peroxidation products such as 4-hydroxy-trans-2-nonenal (HNE and acrolein), chelate metals, and protect against myocardial ischemic injury. Nevertheless, it is unclear whether these peptides protect against cardiac injury by directly reacting with lipid peroxidation products. Hence, to examine whether changes in the structure of carnosine could affect its aldehyde reactivity and metal chelating ability, we synthesized methylated analogs of carnosine, balenine (β-alanine-Nτ-methylhistidine) and di-methylbalenine (DMB), and measured their aldehyde reactivity and metal chelating properties. We found that methylation of Nτ residue of imidazole ring (balenine) or trimethylation of carnosine backbone at Nτ residue of imidazole ring and terminal amine group dimethyl balenine (DMB) abolishes the ability of these peptides to react with HNE. Incubation of balenine with acrolein resulted in the formation of single product (m/z 297), whereas DMB did not react with acrolein. In comparison with carnosine, balenine exhibited moderate acrolein quenching capacity. The Fe2+chelating ability of balenine was higher than carnosine, whereas DMB lacked chelating capacity. Pretreatment of cardiac myocytes with carnosine increased the mean lifetime of myocytes superfused with HNE or acrolein compared with balenine or DMB. Collectively, these results suggest that carnosine protects cardiac myocytes against HNE and acrolein toxicity by directly reacting with these aldehydes. This reaction involves both the amino group of β-alanyl residue and the imidazole residue of L-histidine. Methylation of these sites prevents or abolishes the aldehyde reactivity of carnosine, alters its metal-chelating property, and diminishes its ability to prevent electrophilic injury.
High turnover tissues continually lose specialized cells that are replaced by stem cell activity. In the adult mammalian epidermis, it is unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit amplifying program but instead protect density in the stem cell layer. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.One sentence summaryHeterogeneity in the epidermal stem cell layer reflects a gradual differentiation program that is uncoupled from the loss of proliferative capacity.
Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations1–4 whose relationship to the complete trajectory of differentiation remains unknown. Here we show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.
Regenerative processes in the mammalian skin require coordinated cell-cell communication. Ca2+ signaling can coordinate tissue-level responses in developing and wounded epithelia in tissue explants and invertebrates. However, its role in the homeostatic, regenerative basal layer of the skin epithelium is unknown due to significant challenges in studying signaling dynamics in a spatially complex tissue context in live mice. Here we combine in vivo imaging of dynamic Ca2+ signaling at the single cell level across thousands of cells with a novel computational approach, Geometric Scattering Trajectory Homology (GSTH). GSTH models Ca2+ as signals over a cell adjacency graph and uses a multi-level wavelet-like transform (called a scattering transform) to extract signaling patterns from our high dimensional in vivo datasets. We discover local Ca2+ signaling patterns are orchestrated so that signals flow in a coordinated and directed manner across the tissue, distinct from topographically uncoordinated Ca2+ signaling in excitatory tissues. Directed Ca2+ signaling is regulated by the major gap junction protein in the epidermal stem cell layer, Connexin 43 (Cx43). Cx43 gap junctions are dissociated as cells progress through the cell cycle out of G1 and play an essential role in the progression of stem cells from G2 towards mitosis. Finally, G2 cells display related signaling patterns and are essential for tissue-level signaling coordination. Together, our results provide insight into how such a ubiquitous signaling pathway regulates highly specific behaviors and outcomes at a tissue-wide level to maintain proper homeostasis.
Skin homeostasis is maintained by stem cells, which must communicate to balance their regenerative behaviors. Yet, how adult stem cells signal across regenerative tissue remains unknown due to challenges in studying signaling dynamics in live mice. We combined live imaging in the mouse basal stem cell layer with machine learning tools to analyze patterns of Ca2+ signaling. We show that basal cells display dynamic intercellular Ca2+ signaling among local neighborhoods. We find that these Ca2+ signals are coordinated across thousands of cells and that this coordination is an emergent property of the stem cell layer. We demonstrate that G2 cells are required to initiate normal levels of Ca2+ signaling, while connexin43 connects basal cells to orchestrate tissue-wide coordination of Ca2+ signaling. Lastly, we find that Ca2+ signaling drives cell cycle progression, revealing a communication feedback loop. This work provides resolution into how stem cells at different cell cycle stages coordinate tissue-wide signaling during epidermal regeneration.
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