EGFR-mediated epidermal stem cell motility drives skin regeneration through COL17A1 proteolysis.

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis-mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration.

Cell motion predicts human epidermal stemness.

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation.

Second harmonic generation reveals collagen fibril remodeling in fibroblast-populated collagen gels.

Remodeling of collagen fibrils is involved in a variety of physiological and pathological processes including development, tissue repair, and metastasis. Fibroblast-populated collagen gel contraction has been employed as a model system to investigate the collagen fibril remodeling within three-dimensional collagen matrices. Research on collagen gel contraction is also important for understanding the mechanism underlying connective tissue repair, and for design considerations for engineered tissues in regenerative medicine. Second harmonic generation (SHG) is a non-linier optical effect by which well-ordered protein assemblies, including collagen fibrils, can be visualized without any labeling, and used for a noninvasive imaging of collagen fibrils in the skin. Here we demonstrate that the remodeling of collagen fibrils in the fibroblast-populated collagen gel can be analyzed by SHG imaging with a multiphoton microscope. Two models of collagen gel contraction (freely versus restrained contraction) were prepared, and orientation of fibroblasts, density, diameter, and distribution of collagen fibrils were examined by multiphoton fluorescent and SHG microscopy. Three-dimensional construction images revealed vertical and horizontal orientation of fibroblasts in freely and restrained gel contraction, respectively. Quantitative analysis indicated that collagen fibrils were accumulated within the gel and assembled into the thicker bundles in freely but not restrained collagen gel contraction. We also found that actomyosin contractility was involved in collagen fibril remodeling. This study elucidates how collagen fibrils are remodeled by fibroblasts in collagen gel contraction, and also proves that SHG microscopy can be used for the investigation of the fibroblast-populated collagen gel.

Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells.

Actin filament dynamics impacts keratinocyte stem cell maintenance.

Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1.

Ectodomain shedding of membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the C-terminal fragment in the cell cycle.

Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane protein (proHB-EGF). The proHB-EGF is shed by specific metalloproteases, releasing the N-terminal fragment into the extracellular space as a soluble growth factor (HB-EGF) and the C-terminal fragment (HB-EGF-C) into the intracellular space, where it prevents transcriptional repression by the promyelocytic leukemia zinc finger protein (PLZF). The goal of the present study was to characterize regulation of proHB-EGF shedding and study its temporal variations in HB-EGF-C localization throughout the cell cycle. Quantitative combination analyses of cell surface proHB-EGF and HB-EGF in conditioned medium showed that proHB-EGF shedding occurred during the G(1) cell cycle phase. Laser scanning cytometry (LSC) revealed that HB-EGF-C was internalized into the cytoplasm during the late G1 phase and accumulated in the nucleus beginning in the S phase. Subsequent nuclear export of PLZF occurred during the late S phase. Further, HB-EGF-C was localized around the centrosome following breakdown of the nuclear envelope and was localized to the interzonal space with chromosome segregation in the late M phase. Temporal variations in HB-EGF localization throughout the cell cycle were also characterized by time-lapse imaging of cells expressing YFP-tagged proHB-EGF, and these results were consistent with those obtained in cytometry studies. These results indicate that proHB-EGF shedding and subsequent HB-EGF-C signaling are related with progression of the cell cycle and may provide a clue to understand the unique biological significance of non-receptor-mediated signaling of proHB-EGF in cell growth.

Transactivation of epidermal growth factor receptor after heparin-binding epidermal growth factor-like growth factor shedding in the migration of prostate cancer cells promoted by bombesin.

BACKGROUND: A pathway consisting of bombesin, G-protein coupling receptors (GPCRs), metalloproteases, pro-heparin-binding epidermal growth factor (proHB-EGF), and epidermal growth factor receptor (EGFR) has been reported in prostate cancer cells. The occurrence of HB-EGF shedding from proHB-EGF in this pathway, however, has not been proven directly. In addition, it is still unclear how much this pathway contributes to the migration of prostate cancer cells. In this study, we tried to directly elucidate HB-EGF shedding in this pathway and to determine its contribution to the migration of prostate cancer cells. METHODS: RT-PCR and indirect immunofluorescence staining for HB-EGF and its receptors, such as EGFR and HER4/erbB4, were performed on PC-3 cells. The influences of bombesin, anti-EGFR neutralizing monoclonal antibody, HB-EGF, and HB-EGF shedding inhibitor on the migration of PC-3 cells were studied by means of in vitro wound assays. The amount of HB-EGF shed from PC-3 cells with alkaline phosphatase-tagged HB-EGF in the presence of bombesin was determined by measuring AP activity. Immunoprecipitations and phosphotyrosine Western blotting were performed to detect EGFR transactivated by bombesin. RESULTS: PC-3 expressed HB-EGF and EGFR, but not HER4/erbB4. PC-3 migrated in the presence of bombesin, but its migration was partly inhibited by the neutralizing antibody against EGFR. PC-3 also migrated in the presence of HB-EGF, but HB-EGF shedding inhibitor partly inhibited this phenomenon. HB-EGF was shed from PC-3 cells in the presence of bombesin, and this shedding was inhibited by HB-EGF shedding inhibitor. In addition, the EGFR on PC-3 was activated in the presence of bombesin and inactivated in the presence of HB-EGF shedding inhibitor. CONCLUSIONS: These results indicated that HB-EGF shedding and the following transactivation of EGFR occurs in this pathway and that this pathway partly contributes to the migration of prostate cancer cells.