scholarly journals A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

2017 ◽  
Author(s):  
Ajay Mishra ◽  
Angela Oliveira Pisco ◽  
Benedicte Oules ◽  
Tony Ly ◽  
Kifayathullah Liakath-Ali ◽  
...  
Keyword(s):  
Spatial Dynamics ◽  
Human Keratinocytes ◽  
Cell Renewal ◽  
Primary Human Keratinocytes ◽  
Network Modelling ◽  
Proteomic Data ◽  
Stem Cell Renewal ◽  
Epidermal Homeostasis

AbstractEpidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h to induce differentiation. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases – including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Their activity is antagonised by concomitant upregulation of DUSP10. Boolean network modelling of phosphatase interactions identifies commitment as an inherently unstable biological switch between the stem and differentiated cell states. Furthermore, phosphatase expression is spatially regulated both in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

scholarly journals A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Ajay Mishra ◽  
Bénédicte Oulès ◽  
Angela Oliveira Pisco ◽  
Tony Ly ◽  
Kifayathullah Liakath-Ali ◽  
...  
Keyword(s):  
Spatial Dynamics ◽  
Dynamic Network ◽  
Human Keratinocytes ◽  
Cell Renewal ◽  
Stable States ◽  
Primary Human Keratinocytes ◽  
Network Modelling ◽  
Epidermal Homeostasis

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

scholarly journals A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

2017 ◽  
Author(s):  
Ajay Mishra ◽  
Angela Oliveira Pisco ◽  
Benedicte Oules ◽  
Tony Ly ◽  
Kifayathullah Liakath-Ali ◽  
...  
Keyword(s):  
Stem Cell ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Cell Renewal ◽  
Stable States ◽  
Primary Human Keratinocytes ◽  
Network Modelling ◽  
Epidermal Homeostasis

AbstractEpidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation1,2. While progress has been made in characterising the stem and differentiated cell compartments3, the transition between the two cell states, termed commitment4, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h. We have previously shown that commitment begins at approximately 4h and differentiation is initiated by 8h5. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases – including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote terminal differentiation by negatively regulating ERK MAPK and positively regulating key API transcription factors. Their activity is antagonised by concomitant upregulation of the anti-commitment phosphatase DUSP10. The phosphatases form a dynamic network of transient positive and negative interactions, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem cell and differentiated cell) via an unstable (committed) state. In addition phosphatase expression is spatially regulated relative to the location of stem cells, both in vivo and in response to topographical cues in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

scholarly journals Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuangshuang Wang ◽  
Hua Qian ◽  
Liwei Zhang ◽  
Panpan Liu ◽  
Dexuan Zhuang ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Activated T Cells ◽  
Ras Pathway ◽  
Ras Family ◽  
Interfering Rna

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

scholarly journals Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1294
Author(s):  
Robert Koban ◽  
Markus Neumann ◽  
Philipp P. Nelson ◽  
Heinz Ellerbrok
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Virus Replication ◽  
3D Culture ◽  
Human Keratinocytes ◽  
Infection Model ◽  
Cowpox Virus ◽  
Primary Human Keratinocytes

Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.

scholarly journals In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chee-Wai Wong ◽  
Catherine F. LeGrand ◽  
Beverley F. Kinnear ◽  
Radoslaw M. Sobota ◽  
Rajkumar Ramalingam ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Collagen I ◽  
Matrix Composition ◽  
Major Influence ◽  
Primary Human Keratinocytes ◽  
Matrix Deposition

AbstractThe long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

scholarly journals Expression of membrane type 1 matrix metalloproteinase in papillomavirus-positive cells: role of the human papillomavirus (HPV) 16 and HPV8 E7 gene products

2005 ◽  
Vol 86 (5) ◽  
pp. 1291-1296 ◽  
Author(s):  
Sigrun Smola-Hess ◽  
Jenny Pahne ◽  
Cornelia Mauch ◽  
Paola Zigrino ◽  
Hans Smola ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cellular Proliferation ◽  
Human Keratinocytes ◽  
Hpv Infection ◽  
Host Cells ◽  
Primary Human Keratinocytes ◽  
E7 Gene ◽  
Membrane Type

Matrix metalloproteinases (MMPs) degrade extracellular matrix. They are involved in cellular proliferation, migration, angiogenesis, invasion and metastasis. MT-1 MMP, a membrane-bound MMP, is expressed in carcinomas of the uterine cervix in vivo. This type of cancer is associated with human papillomavirus (HPV) infection. Here it was shown that keratinocytes transformed with HPV16 or HPV18 in vitro, and HPV-positive cervical carcinoma cell lines, constitutively expressed MT-1 MMP. Expression of the E7 protein from the mucosal and cutaneous high-risk types HPV16 and HPV8, but not from the cutaneous low-risk type HPV1, was sufficient to induce MT-1 MMP expression in primary human keratinocytes and HaCaT cells. As a consequence, MMP-2 was activated. MT-1 MMP expression might play a role in the HPV life cycle by promoting proliferation of host cells and might contribute to their invasive phenotype during malignant progression.

scholarly journals In VivoApplicability ofNeosartorya fischeriAntifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis

2018 ◽  
Vol 63 (2) ◽  
pp. e01777-18 ◽  
Author(s):  
Renátó Kovács ◽  
Jeanett Holzknecht ◽  
Zoltán Hargitai ◽  
Csaba Papp ◽  
Attila Farkas ◽  
...  
Keyword(s):  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Vulvovaginal Candidiasis ◽  
Antifungal Protein ◽  
Primary Human Keratinocytes ◽  
Fungal Cell ◽  
Efficient Treatment ◽  
Content Type

ABSTRACTAs a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-formingCandidaspecies, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of severalCandidaspp.in vitro; however, no information is available about theirin vivoantifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis,Neosartorya fischeriantifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistantCandida albicansstrain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilmin vitro. We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MICin vitro. In vivomurine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistantC. albicanscells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

scholarly journals In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

2018 ◽  
Author(s):  
Chee-Wai Wong ◽  
Beverley F. Kinnear ◽  
Radoslaw M. Sobota ◽  
Rajkumar Ramalingam ◽  
Catherine F. LeGrand ◽  
...  
Keyword(s):  
Cell Fate ◽  
Dermal Fibroblast ◽  
Human Keratinocytes ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Primary Human Keratinocytes ◽  
Matrix Deposition ◽  
Forming Efficiency

SummaryThe long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the dermal fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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