Niche stiffening compromises hair follicle stem cell potential during ageing by reducing bivalent promoter accessibility.

Tissue turnover requires activation and lineage commitment of tissue-resident stem cells (SCs). These processes are impacted by ageing, but the mechanisms remain unclear. Here, we addressed the mechanisms of ageing in murine hair follicle SCs (HFSCs) and observed a widespread reduction in chromatin accessibility in aged HFSCs, particularly at key self-renewal and differentiation genes, characterized by bivalent promoters occupied by active and repressive chromatin marks. Consistent with this, aged HFSCs showed reduced ability to activate bivalent genes for efficient self-renewal and differentiation. These defects were niche dependent as the transplantation of aged HFSCs into young recipients or synthetic niches restored SC functions. Mechanistically, the aged HFSC niche displayed widespread alterations in extracellular matrix composition and mechanics, resulting in mechanical stress and concomitant transcriptional repression to silence promoters. As a consequence, increasing basement membrane stiffness recapitulated age-related SC changes. These data identify niche mechanics as a central regulator of chromatin state, which, when altered, leads to age-dependent SC exhaustion.

Transepidermal UV radiation of scalp skin ex vivo induces hair follicle damage that is alleviated by the topical treatment with caffeine.

OBJECTIVES: Although the effect of ultraviolet radiation (UVR) on human skin has been extensively studied, very little is known on how UVR impacts on hair follicle (HF) homeostasis. Here, we investigated how solar spectrum UVR that hits the human skin surface impacts on HF biology, and whether any detrimental effects can be mitigated by a widely used cosmetic and nutraceutical ingredient, caffeine. METHODS: Human scalp skin with terminal HFs was irradiated transepidermally ex vivo using either 10 J/cm2 UVA (340-440 nm) + 20 mJ/cm2 UVB (290-320 nm) (low dose) or 50 J/cm2 UVA + 50 mJ/cm2 UVB (high dose) and organ-cultured under serum-free conditions for 1 or 3 days. 0.1% caffeine (5.15 mmol/L) was topically applied for 3 days prior to UV exposure with 40 J/cm2 UVA + 40 mJ/cm2 UVB and for 3 days after UVR. The effects on various toxicity and vitality read-out parameters were measured in defined skin and HF compartments. RESULTS: Consistent with previous results, transepidermal UVR exerted skin cytotoxicity and epidermal damage. Treatment with high and/or low UVA+UVB doses also induced oxidative DNA damage and cytotoxicity in human HFs. In addition, it decreased proliferation and promoted apoptosis of HF outer root sheath (ORS) and hair matrix (HM) keratinocytes, stimulated catagen development, differentially regulated the expression of HF growth factors, and induced perifollicular mast cell degranulation. UVR-mediated HF damage was more severe after irradiation with high UVR dose and reached also proximal HF compartments. The topical application of 0.1% caffeine did not induce skin or HF cytotoxicity and stimulated the expression of IGF-1 in the proximal HF ORS. However, it promoted keratinocyte apoptosis in selected HF compartments. Moreover, caffeine provided protection towards UVR-mediated HF cytotoxicity and dystrophy, keratinocyte apoptosis, and tendential up-regulation of the catagen-promoting growth factor. CONCLUSION: Our study highlights the clinical relevance of our scalp UV irradiation ex vivo assay and provides the first evidence that transepidermal UV radiation negatively affects important human HF functions. This suggests that it is a sensible prophylactic strategy to integrate agents such as caffeine that can act as HF photoprotectants into sun-protective cosmeceutical and nutraceutical formulations.

OBJECTIFS: Alors que l'effet de rayons ultraviolets (RUV) sur la peau humaine a été largement étudié, on sait très peu de choses de l'impact des UV sur l'homéostasie du follicule pileux (FP). Ici, nous avons étudié l'effet du spectre des RUV solaires qui atteignent la surface de la peau humaine sur la biologie du FP, et si tout effet nocif peut être atténué par de la caféine, un ingrédient cosmétique et neutraceutique largement utilisé. MÉTHODES: Une peau de cuir chevelu humain avec ses FP terminaux a été irradiée ex vivo via l'épiderme soit par 10 J/cm2 d'UVA (340-440 nm) + 20 mJ/cm2 d'UVB (290-320 nm) (dose faible) soit par 50 J/cm2 d'UVA + 50 mJ/cm2 d'UVB (dose élevée) et placée en culture sans sérum pendant 1 ou 3 jours. 0,1% (5,15 mM) de caféine a été appliquée par voie topique pendant 3 jours avant l'exposition aux UV à raison de 40 J/cm2 d'UVA + 40 mJ/cm2 UVB et pendant 3 jours après l'exposition aux RUV. Les effets sur divers paramètres de toxicité et de vitalité ont été mesurés au niveau de compartiments définis de la peau et des FP. RÉSULTATS: Cohérent avec les résultats précédents, les RUV transépidermique ont exercé une cytotoxicité au niveau de la peau et des lésions épidermiques. Le traitement par des doses élevées et/ou faibles d'UVA+UVB a également induit des lésions oxydatives de l'ADN et une cytotoxicité au niveau des FP humains. En outre, il a diminué la prolifération et favorisé l'apoptose de la gaine externe de la racine (ORS) du FP et des kératinocytes de la matrice des cheveux (MC), a stimulé le développement de la phase catagène, a régulé de manière différentielle l'expression des facteurs de croissance des FP, et induit une dégranulation périfolliculaire des mastocytes. Les lésions du FP médiées par les RUV étaient plus graves après une irradiation par dose élevée de RUV et atteignaient également les compartiments proximaux du FP. L'application topique de 0,1 % de caféine n'a pas induit de cytotoxicité de la peau ou du FP et a stimulé l'expression d'IGF-1 dans la partie proximale de l'ORS du FP. Cependant, elle a promu l'apoptose des kératinocytes dans certains compartiments de FP. En outre, la caféine a fourni une protection des FP contre la cytotoxicité et la dystrophie médiées par les RUV, l'apoptose des kératinocytes et une régulation à tendance positive de l'effet catagène induit par le facteur de croissance. CONCLUSION: Notre étude souligne la pertinence clinique de notre dosage d'irradiation UV ex vivo du cuir chevelu et fournit la première preuve que le rayonnement UV transépidermique affecte négativement d'importantes fonctions du FP chez l'homme. Cela suggère que l'intégration d'agents photoprotecteurs des FP tels que la caféine dans les formulations cosmétiques et nutraceutiques des écrans solaires pourrait constituer une stratégie prophylactique sensée.

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators.

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.

Role of Integrins α1ß1 and α2ß1 in Wound and Tumor Angiogenesis in Mice.

Integrins are transmembrane receptors composed of one α subunit and one ß subunit and are involved in cellular growth, differentiation, and apoptosis. The collagen-binding integrins α1ß1 and α2ß1 have been shown to regulate wound and tumor vascularization by different mechanisms. In this study, we assessed wound and tumor vascularization in mice with genetic ablation of both integrin subunits α1 and α2, which resulted in loss of integrins α1ß1 and α2ß1. Wound angiogenesis was investigated in excisional wounds that were inflicted on the back skin of control and mice lacking integrin α1ß1 and α2ß1. Mutant mice displayed reduced wound angiogenesis, which correlated with decreased macrophage numbers at 3 and 7 days after injury, and showed significantly attenuated vascularization of sponge implants. Angiogenesis induced by tumors arising from intradermal injection of B16 F1 melanoma cells was also reduced in comparison to controls 7 days after injection. This reduction in angiogenesis correlated with increased levels and activity of circulating matrix metalloproteinase 9 and elevated angiostatin levels in plasma of mutant mice, which reduced endothelial cell proliferation. Ex vivo mutant aortic ring explants developed significantly fewer and thinner aortic sprouts with fewer branch points than controls because of impaired endothelial cell proliferation. In conclusion, the loss of integrins α1ß1 and α2ß1 in mice results in reduced wound and tumor angiogenesis by cell-autonomous and extrinsic mechanisms.

Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment.

Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.

Integrin-linked kinase regulates the niche of quiescent epidermal stem cells.

Stem cells reside in specialized niches that are critical for their function. Quiescent hair follicle stem cells (HFSCs) are confined within the bulge niche, but how the molecular composition of the niche regulates stem cell behaviour is poorly understood. Here we show that integrin-linked kinase (ILK) is a key regulator of the bulge extracellular matrix microenvironment, thereby governing the activation and maintenance of HFSCs. ILK mediates deposition of inverse laminin (LN)-332 and LN-511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise BM composition tunes activities of Wnt and transforming growth factor-ß pathways and subsequently regulates HFSC activation. Notably, reconstituting an optimal LN microenvironment restores the altered signalling in ILK-deficient cells. Aberrant stem cell activation in ILK-deficient epidermis leads to increased replicative stress, predisposing the tissue to carcinogenesis. Overall, our findings uncover a critical role for the BM niche in regulating stem cell activation and thereby skin homeostasis.

ILK: a pseudokinase with a unique function in the integrin-actin linkage.

ILK (integrin-linked kinase) is a central component of cell-matrix adhesions and an important regulator of integrin function. It forms a ternary complex with two other adaptor proteins, PINCH (particularly interesting cysteine- and histidine-rich protein) and parvin, forming the IPP (ILK-PINCH-parvin) complex that regulates the integrin-actin linkage as well as microtubule dynamics. These functions are essential for processes such as cell migration and matrix remodelling. The present review discusses the recent advances on the structural and functional characterization of ILK and the long-standing debate regarding its reported kinase activity.