Xinyue Wang

Stabilizing mutations of KLHL24 ubiquitin ligase cause loss of keratin 14 and human skin fragility

Skin integrity is essential for protection from external stress and trauma. Defects in structural proteins such as keratins cause skin fragility, epitomized by epidermolysis bullosa (EB), a life-threatening disorder. Here we show that dominant mutations of KLHL24, encoding a cullin 3-RBX1 ubiquitin ligase substrate receptor, cause EB. We have identified start-codon mutations in the KLHL24 gene in five patients with EB. These mutations lead to truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-ΔN28). KLHL24-ΔN28 is more stable than its wild-type counterpart owing to abolished autoubiquitination. We have further identified keratin 14 (KRT14) as a KLHL24 substrate and found that KLHL24-ΔN28 induces excessive ubiquitination and degradation of KRT14. Using a knock-in mouse model, we have confirmed that the Klhl24 mutations lead to stabilized Klhl24-ΔN28 and cause Krt14 degradation. Our findings identify a new disease-causing mechanism due to dysregulation of autoubiquitination and open new avenues for the treatment of related disorders.

Estrogen Leads to Reversible Hair Cycle Retardation through Inducing Premature Catagen and Maintaining Telogen

Estrogen dysregulation causes hair disorder. Clinical observations have demonstrated that estrogen raises the telogen/anagen ratio and inhibits hair shaft elongation of female scalp hair follicles. In spite of these clinical insights, the properties of estrogen on hair follicles are poorly dissected. In the present study, we show that estrogen induced apoptosis of precortex cells and caused premature catagen by up-regulation of TGF β2. Immediately after the premature catagen, the expression of anagen chalone BMP4 increased. The up-regulation of BMP4 may further function to prevent anagen transition and maintain telogen. Interestingly, the hair follicle stem cell niche was not destructed during these drastic structural changes caused by estrogen. Additionally, dermal papilla cells, the estrogen target cells in hair follicles, kept their signature gene expressions as well as their hair inductive potential after estrogen treatment. Retention of the characteristics of both hair follicle stem cells and dermal papilla cells determined the reversibility of the hair cycle suppression. These results indicated that estrogen causes reversible hair cycle retardation by inducing premature catagen and maintaining telogen.

mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration

Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration.

GPR39 marks specific cells within the sebaceous gland and contributes to skin wound healing

G protein-coupled receptors (GPCRs) mediate multiple key biological processes in the body. The orphan receptor GPR39 has been reported to be involved in various pathophysiological events. However, the function of GPR39 in skin biology remains unknown. Using a genetically engineered mouse strain in which lacZ expression faithfully replaced endogenous Gpr39 expression, we discovered a unique expression pattern of Gpr39 in the sebaceous gland (SG). Using various methods, we confirmed that GPR39 marked a specific cell population at the opening of the SG and colocalised with the SG stem cell marker Blimp1. Further investigations showed that GPR39 was spatiotemporally expressed during skin wound repair. Although it was dispensable for skin development and homeostasis, GPR39 contributed positively to skin wound healing: its loss led to a delay in wound healing during the intermediate stage. The present study reveals a novel role of GPR39 in both dermatology and stem cell biology that has not been previously recognised.

Senescence of human skin-derived precursors regulated by Akt-FOXO3-p27KIP1/p15INK4b signaling

Multipotent skin-derived precursors (SKPs) are dermal stem cells with the capacity to reconstitute the dermis and other tissues, such as muscles and the nervous system. Thus, the easily available human SKPs (hSKPs) hold great promises in regenerative medicine. However, long-term expansion is difficult for hSKPs in vitro. We previously demonstrated that hSKPs senesced quickly under routine culture conditions. To identify the underlying mechanisms so as to find an effective way to expand hSKPs, time-dependent microarray analysis of gene expression in hSKPs during in vitro culture was performed. We found that the senescence of hSKPs had a unique gene expression pattern that differs from reported typical senescence. Subsequent investigation ruled out the role of DNA damage and classical p53 and p16INK4a signaling in hSKP senescence. Examination of cyclin-dependent kinase inhibitors revealed the involvement of p15INK4b and p27KIP1. Further exploration about upstream signals indicated the contribution of Akt hypo-activity and FOXO3 to hSKP senescence. Forced activation of Akt and knockdown of FOXO3, p15INK4b and p27KIP1 effectively inhibited hSKP senescence and promoted hSKP proliferation. The unique senescent phenotype of human dermal stem cells and the role of Akt-FOXO3-p27KIP1/p15INK4b signaling in regulating hSKP senescence provide novel insights into the senescence and self-renewal regulation of adult stem cells. The present study also points out a way to propagate hSKPs in vitro so as to fulfill their promises in regenerative medicine.

Rotary Suspension Culture Enhances Mesendoderm Differentiation of Embryonic Stem Cells Through Modulation of Wnt/β-catenin Pathway

Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.

Spatiotemporal Expression of p63 in Mouse Epidermal Commitment

The embryonic surface ectoderm is a simple flat epithelium consisting of cells that express the cytokeratins K8/K18. Before stratification, K5/K14 expression substitutes K8/K18 expression, marking the event called epidermal commitment. Previous studies show that the transcription factor p63 plays an essential role in epidermal commitment. However, detailed expression information of p63 during early epidermal development in mice is still unclear. We systematically studied the expression pattern of p63 in mouse epidermal commitment, together with K8 and K5. We show that p63 expression could be detected as early as E8.5 in mouse embryos preceding epidermal commitment. p63 expression first appears near the newly formed somites and the posterior part of the embryo, further expanding to the whole embryonic surface with particular enrichment in the first branchial arches and the limb buds. ΔNp63 is the major class of isoforms expressed in this period. Relative expression intensity of p63 depends on the embryonic position. In summary, there is a sequential and regular expression pattern of K8, p63 and K5 in mouse epidermal commitment. Our study not only contributes to understanding the early events during epidermal development but also provides a basal tool to study the function of p63 in mammals.