Temperature impacts human epidermal stem cell behaviour through thermoTRPs and mTOR signalling

Adult stem cells are characterized by their unique ability to self-renew and give rise to a progeny of specialized/differentiated cells. Therefore, they have a remarkable potential for regenerative medicine. However, the use of adult stem cells is restrained by several obstacles such as limited in vitro amplification and uncontrolled differentiation. Adult stem cells reside in tightly regulated microenvironments termed “niches”. The niche is composed of nursing cells, extracellular matrix and a variety of signalling factors that dynamically regulate stem cell behaviour. Consequently, recapitulating the niche in vitro is fundamental in manipulating adult stem cells. In recent years, the physicochemical microenvironment of the niche (e.g. temperature, pH, oxygen tension) has proven to be as important as classical signalling pathways for the regulation of stem cell fate. Therefore, the aim of this thesis is to understand the influence of temperature variations on human epidermal stem cell behaviour. This effort has been accomplished in three main directions. First, we have contributed to the development of two different telemetric temperature sensors that precisely measure and record temperature in vivo and in vitro. These sensors have allowed us to demonstrate that epidermal keratinocytes are continuously subjected to small physiological temperature variations (3 ± 2oC) in vivo. Then, we have engineered a device that allows a tight and dynamic control of temperature while simultaneously providing real-time imaging of cultured human epidermal stem cells. It is shown that temperature variations of 1oC are not trivial, since they significantly affect human epidermal stem cell growth and proliferation. Second, we have shown that human keratinocytes sense temperature by means of temperature-sensitive calcium channels termed thermoTRPs (Transient Receptor Potential). Consequently, we have monitored intracellular calcium concentrations to evaluate the reaction of epidermal stem cells to thermal or chemical stimulations. The strongest calcium influxes have been observed after temperature drops (from 37 to 32oC) suggesting that human keratinocytes are particularly sensitive to temperature decreases. Furthermore, we have demonstrated the importance of these channels for keratinocyte viability by silencing a thermoTRP channel (i.e. TRPV3) using lentiviral delivery of shRNA. Third, we have demonstrated that thermal signals affecting keratinocytes are integrated through the mTOR (mammalian Target Of Rapamycin) signalling pathway, since cool stimuli (32oC) decreased mTOR complex 1 (mTORC1) kinase activity. Culture at 32oC and 100nm rapamycin treatment affected growth and proliferation of human keratinocyte stem cells similarly, further stressing the inhibitory effects of cool temperatures on mTORC1. We also have observed that inhibition of mTORC1 induced nuclear translocation of mTOR, suggesting a putative transcriptional role for mTOR. To conclude, we have established that prolonged inhibition of mTORC1 favours an apparent maintenance of the stem cell phenotype by reducing clonal conversion, without however stopping stem cell aging. Altogether, our results demonstrate that thermoTRPs are wired to the mTOR signalling in human epidermal stem cells. Furthermore, our work thoroughly supports the hypothesis that temperature is a significant actor of the epidermal stem cell niche as it can directly affect stem cell behaviour.

Identification and functional characterization of protein kinases that modulate Notch signaling under physiological conditions and in cancer

Chhavi Jain

The Notch signaling pathway is a key regulator of cell fate decisions in embryonic development and in adult tissue homeostasis. Mounting evidence suggests that Notch signaling is frequently deregulated in human neoplasms, where depending upon the cellular context it can function both as an oncogene or a tumor suppressor. A plethora of studies shed light on how Notch crosstalks with signaling pathways downstream to manifest either its oncogenic or tumor suppressive activity. However, regulatory networks upstream of the Notch signaling pathway are still poorly understood. Since protein kinases are well-known regulators of a majority of cell signaling pathways, the aim of the current study was to identify novel positive and negative regulatory kinases that modulate Notch signaling. Using a HeLa cell based co-culture assay to read Notch-dependent luciferase activity, a small interference RNA (siRNA) library against the human kinase genome was previously tested in a high-throughput manner. Validation of top candidate kinases from the screening using both siRNA as well as pharmacological inhibitors led to further elimination of false positives and identification of Protein Kinase B (AKT2) and Calmodulin Kinase II (CaMKII) as key positive while Janus kinases (JAKs) as key negative regulators of the Notch signaling pathway. In the first part of the study, we analyzed the positive regulation of AKT2 and CaMKII kinases on Notch signaling in the T-cell acute lymphoblastic leukemia (T-ALL) cells where Notch is oncogenic. It was shown that pharmacological inhibition of either AKT2 or CaMKII kinase in T-ALL cell lines in vitro abrogated the expression of active Notch1 intracellular domain (N1-ICD) as well as of Notch target genes. Moreover, inhibition of these kinases blocked proliferation of T-ALL cells. In the second part of the study, we analyzed the negative regulation of JAK kinases on Notch signaling using a distinct physiological context where Notch is tumor suppressive. It was shown that pharmacological inhibition of JAK kinases led to an induction of Notch receptor transcription and of Notch target genes in the human primary epidermal keratinocytes (HPEK) as well as in the skin squamous cell carcinoma (SCC) cell lines. In addition, the pharmacological inhibition of JAK kinases induced a block in proliferation, cell cycle arrest and differentiation in HPEKs and skin SCCs by increased expression of early differentiation markers. Suppression of Notch signaling in primary keratinocytes counteracted the differentiation-inducing effect of JAK inhibition. Since JAK inhibition affected Notch transcription, global gene expression profiling using RNA sequencing was done to identify transcription factors involved in an indirect regulation of JAK kinases on the Notch cascade. EGR1 and EGR2 belonging to the early growth response family of transcription factors were significantly modulated downstream of JAK inhibition. In vivo, topical inhibition of JAK kinases provided marked resistance against chemically induced cutaneous carcinogenesis.Taken together, our data reveals a key role of AKT2 and CaMKII kinases in positive regulation while of JAK kinases in the negative regulation of Notch signaling, of potential relevance for combinatory approaches for cancer therapy. Pharmacological inhibitors against these kinases could be tested for their ability to modulate Notch signaling and may prove highly beneficial in the therapy of Notch-driven cancers.

EPFL2016

Early Fate Choice Determines in vivo Engraftment and in vitro Behavior of Epidermal Stem Cells

François Gorostidi

Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demon- strate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo ampli- fication of stem cells, the two limiting steps of cell replacement therapy.

EPFL2012

Inhibition of Fibroblast Growth by Notch1 Signaling Is Mediated by Induction of Wnt11-Dependent WISP-1

Yan Li, Freddy Radtke, Yurong Tan, Jialin Zhang

Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox)) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1(-/-)) MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox) MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

Public Library of Science2012