Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence.

Chronic, sustained exposure to stressors can profoundly affect tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here we report that the stress hormone corticosterone-which is derived from the adrenal gland and is the rodent equivalent of cortisol in humans-regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. In the absence of systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, increased levels of corticosterone prolong HFSC quiescence and maintain hair follicles in an extended resting phase. Mechanistically, corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term.

Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2.

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.

Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway.

The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both ß-arrestin 1 and ß-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s.

Loss of Cxcl12/Sdf-1 in adult mice decreases the quiescent state of hematopoietic stem/progenitor cells and alters the pattern of hematopoietic regeneration after myelosuppression.

The C-X-C-type chemokine Cxcl12, also known as stromal cell-derived factor-1, plays a critical role in hematopoiesis during fetal development. However, the functional requirement of Cxcl12 in the adult hematopoietic stem/progenitor cell (HSPC) regulation was still unclear. In this report, we developed a murine Cxcl12 conditional deletion model in which the target gene can be deleted at the adult stage. We found that loss of stroma-secreted Cxcl12 in the adult led to expansion of the HSPC population as well as a reduction in long-term quiescent stem cells. In Cxcl12-deficient bone marrow, HSPCs were absent along the endosteal surface, and blood cell regeneration occurred predominantly in the perisinusoidal space after 5-fluorouracil myelosuppression challenge. Our results indicate that Cxcl12 is required for HSPC homeostasis regulation and is an important factor for osteoblastic niche organization in adult stage bone marrow.

Inhibition of PIKfyve kinase prevents infection by Zaire ebolavirus and SARS-CoV-2.

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric VSV containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or SARS-CoV-2 (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small molecule inhibitors of the main endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define new tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.

Caenorhabditis elegans reveals a FxNPxY-independent low-density lipoprotein receptor internalization mechanism mediated by epsin1.

Low-density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNA interference screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to the reduction observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR.

Ectopic expression of herpes simplex virus-thymidine kinase gene in human non-small cell lung cancer cells conferred caspase-activated apoptosis sensitized by ganciclovir.

Human non-small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV-TK in stable transfectants. Development in the HSV-tk/GCV system toward cell death was initiated with cell-cycle accumulation at S and G(2)/M phases, immediately followed by the appearance of sub-G(0)/G(1) cells after drug exposure. To investigate the regulation of cell-cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B(1) before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV-tk gene. Our results demonstrate that the HSV-tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development.

A novel PEPP homeobox gene, TOX, is highly glutamic acid rich and specifically expressed in murine testis and ovary.

The homeobox gene superfamily has been highly conserved throughout evolution. These genes act as transcription factors during several important developmental processes. To explore the functional roles of homeobox genes in spermatogenesis, we performed a degenerate oligonucleotide polymerase chain reaction (PCR) screening of a testis cDNA library and isolated a novel mouse homeobox gene. This gene, which we named Tox, encodes a homeodomain protein distantly related to members of the Paired/Pax (Prd/Pax) family. A phylogenetic analysis revealed Tox to be a member of the recently defined PEPP subfamily of Paired-like homeobox genes. Tox was mapped to chromosome X, with its homeodomain organized into three exons. A special feature of Tox is that the encoded protein sequence contains two poly-glutamic acid (poly E) stretches, which make Tox highly acidic. Tox transcripts were detected predominately in the testis and ovary of mice. Tox expression in testes was initiated soon after birth, mainly in Sertoli cells and spermatogonia; however, in adult mice, Tox expression shifts to the spermatids and spermatozoa. Tox expression in ovaries was detected in somatic cells of follicles, early on in theca cells, and in both granulosa and theca cells at the later stages of follicular development. Based on these results, Tox may play an important role during gametogenesis.