Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host.

Epidermal Notch signalling: differentiation, cancer and adhesion.

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

Confluence switch signaling regulates ECM composition and the plasmin proteolytic cascade in keratinocytes.

In culture, cell confluence generates signals that commit actively growing keratinocytes to exit the cell cycle and differentiate to form a stratified epithelium. Using a comparative proteomic approach, we studied this 'confluence switch' and identified a new pathway triggered by cell confluence that regulates basement membrane (BM) protein composition by suppressing the uPA-uPAR-plasmin pathway. Indeed, confluence triggers adherens junction maturation and enhances TGF-ß and activin A activity, resulting in increased deposition of PAI-1 and perlecan in the BM. Extracellular matrix (ECM)-accumulated PAI-1 suppresses the uPA-uPAR-plasmin pathway and further enhances perlecan deposition by inhibiting its plasmin-dependent proteolysis. We show that perlecan deposition in the ECM strengthens cell adhesion, inhibits keratinocyte motility and promotes additional accumulation of PAI-1 in the ECM at confluence. In agreement, during wound-healing, perlecan concentrates at the wound-margin, where BM matures to stabilize keratinocyte adhesion. Our results demonstrate that confluence-dependent signaling orchestrates not only growth inhibition and differentiation, but also controls ECM proteolysis and BM formation. These data suggest that uncontrolled integration of confluence-dependent signaling, might favor skin disorders, including tumorigenesis, not only by promoting cell hyperproliferation, but also by altering protease activity and deposition of ECM components.

ECM and epithelial stem cells: the scaffold of destiny.

Adult stem cells play a critical role in maintaining tissue homeostasis and promoting longevity. The intricate organization and presence of common markers among adult epithelial stem cells in the intestine, lung, and skin serve as hallmarks of these cells. The specific location pattern of these cells within their respective organs highlights the significance of the niche in which they reside. The extracellular matrix (ECM) not only provides physical support but also acts as a reservoir for various biochemical and biophysical signals. We will consider differences in proliferation, repair, and regenerative capacities of the three epithelia and review how environmental cues emerging from the niche regulate cell fate. These cues are transduced via mechanosignaling, regulating gene expression, and bring us to the concept of the fate scaffold. Understanding both the analogies and discrepancies in the mechanisms that govern stem cell fate in various organs can offer valuable insights for rejuvenation therapy and tissue engineering.

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases.

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.

Laminin-binding integrins induce Dll4 expression and Notch signaling in endothelial cells.

RATIONALE: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. OBJECTIVE: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. METHODS AND RESULTS: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2ß1 and α6ß1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2ß1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. CONCLUSIONS: We show that, in endothelial cells, ligation of α2ß1 and α6ß1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin ß1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Transcription factor E4F1 is essential for epidermal stem cell maintenance and skin homeostasis.

A growing body of evidence suggests that the multifunctional protein E4F1 is involved in signaling pathways that play essential roles during normal development and tumorigenesis. We generated E4F1 conditional knockout mice to address E4F1 functions in vivo in newborn and adult skin. E4F1 inactivation in the entire skin or in the basal compartment of the epidermis induces skin homeostasis defects, as evidenced by transient hyperplasia in the interfollicular epithelium and alteration of keratinocyte differentiation, followed by loss of cellularity in the epidermis and severe skin ulcerations. E4F1 depletion alters clonogenic activity of epidermal stem cells (ESCs) ex vivo and ends in exhaustion of the ESC pool in vivo, indicating that the lesions observed in the E4F1 mutant skin result, at least in part, from cell-autonomous alterations in ESC maintenance. The clonogenic potential of E4F1 KO ESCs is rescued by Bmi1 overexpression or by Ink4a/Arf or p53 depletion. Skin phenotype of E4F1 KO mice is also delayed in animals with Ink4a/Arf and E4F1 compound gene deficiencies. Our data identify a regulatory axis essential for ESC-dependent skin homeostasis implicating E4F1 and the Bmi1-Arf-p53 pathway.

alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation.

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies.

Tracing the cellular dynamics of sebaceous gland development in normal and perturbed states.

The sebaceous gland (SG) is an essential component of the skin, and SG dysfunction is debilitating1,2. Yet, the cellular bases for its origin, development and subsequent maintenance remain poorly understood. Here, we apply large-scale quantitative fate mapping to define the patterns of cell fate behaviour during SG development and maintenance. We show that the SG develops from a defined number of lineage-restricted progenitors that undergo a programme of independent and stochastic cell fate decisions. Following an expansion phase, equipotent progenitors transition into a phase of homeostatic turnover, which is correlated with changes in the mechanical properties of the stroma and spatial restrictions on gland size. Expression of the oncogene KrasG12D results in a release from these constraints and unbridled gland expansion. Quantitative clonal fate analysis reveals that, during this phase, the primary effect of the Kras oncogene is to drive a constant fate bias with little effect on cell division rates. These findings provide insight into the developmental programme of the SG, as well as the mechanisms that drive tumour progression and gland dysfunction.

Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy.

Dysregulation of extracellular matrix (ECM) deposition and cellular metabolism promotes tumor aggressiveness by sustaining the activity of key growth, invasion, and survival pathways. Yet mechanisms by which biophysical properties of ECM relate to metabolic processes and tumor progression remain undefined. In both cancer cells and carcinoma-associated fibroblasts (CAFs), we found that ECM stiffening mechanoactivates glycolysis and glutamine metabolism and thus coordinates non-essential amino acid flux within the tumor niche. Specifically, we demonstrate a metabolic crosstalk between CAF and cancer cells in which CAF-derived aspartate sustains cancer cell proliferation, while cancer cell-derived glutamate balances the redox state of CAFs to promote ECM remodeling. Collectively, our findings link mechanical stimuli to dysregulated tumor metabolism and thereby highlight a new metabolic network within tumors in which diverse fuel sources are used to promote growth and aggressiveness. Furthermore, this study identifies potential metabolic drug targets for therapeutic development in cancer.

Cell metabolism regulates integrin mechanosensing via an SLC3A2-dependent sphingolipid biosynthesis pathway.

Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.

Dermal Fibroblast SLC3A2 Deficiency Leads to Premature Aging and Loss of Epithelial Homeostasis.

Skin homeostasis relies on fine-tuning of epidermis-dermis interactions and is affected by aging. While extracellular matrix (ECM) proteins, such as integrins, are involved in aging, the molecular basis of the skin changes needs to be investigated further. Here, we showed that integrin co-receptor, SLC3A2, required for cell proliferation, is expressed at the surface of resting dermal fibroblasts in young patients and is reduced drastically with aging. In vivo SLC3A2 dermal fibroblast deletion induced major skin phenotypes resembling premature aging. Knockout mice (3 months old) presented strong defects in skin elasticity due to altered ECM assembly, which impairs epidermal homeostasis. SLC3A2 dermal fibroblast loss led to an age-associated secretome profile, with 77% of identified proteins belonging to ECM and ECM-associated proteins. ECM not only contributes to skin mechanical properties, but it is also a reservoir of growth factors and bioactive molecules. We demonstrate that dermal fibroblast SLC3A2 is required for ECM to fully exert its structural and reservoir role allowing proper and efficient TGF-ß localization and activation. We identified SLC3A2 as a protective controller of dermal ECM stiffness and quality required to maintain the epidermis to dermis interface as functional and dynamic.

The Human Rho-GEF trio and its target GTPase RhoG are involved in the NGF pathway, leading to neurite outgrowth.

Rho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange. Trio displays two Rho-GEF domains, GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA, and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated. Here, we show that human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, we demonstrate that Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, we show that Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling.

The body's tailored suit: Skin as a mechanical interface.

Skin, by nature, is very similar to the Rouquayrol-Denayrouze suit mentioned by Jules Verne in Twenty Thousand Leagues Under the Sea: it allows "to risk (…) new physiological conditions without suffering any organic disorder". Mechanical cues, to the same extent as other environmental parameters, are such "new physiological conditions". Indeed, skin's primary function is to form a protective barrier to shield inner tissues from the external environment. This requires unique mechanical properties as well as the ability to sense mechanical cues from the environment in order to prevent or repair mechanical damages as well as to function as the primary mechanosensory interface of the whole body.

CD98hc (SLC3A2) loss protects against ras-driven tumorigenesis by modulating integrin-mediated mechanotransduction.

CD98hc (SLC3A2) is the heavy chain component of the dimeric transmembrane glycoprotein CD98, which comprises the large neutral amino acid transporter LAT1 (SLC7A5) in cells. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor prognosis clinically, but its exact contributions to tumorigenesis are uncertain. In this study, we showed that genetic deficiency of CD98hc protects against Ras-driven skin carcinogenesis. Deleting CD98hc after tumor induction was also sufficient to cause regression of existing tumors. Investigations into the basis for these effects defined two new functions of CD98hc that contribute to epithelial cancer beyond an intrinsic effect of CD98hc on tumor cell proliferation. First, CD98hc increased the stiffness of the tumor microenvironment. Second, CD98hc amplified the capacity of cells to respond to matrix rigidity, an essential factor in tumor development. Mechanistically, CD98hc mediated this stiffness sensing by increasing Rho kinase (ROCK) activity, resulting in increased transcription mediated by YAP/TAZ, a nuclear relay for mechanical signals. Our results suggest that CD98hc contributes to carcinogenesis by amplifying a positive feedback loop, which increases both extracellular matrix stiffness and resulting cellular responses. This work supports a rationale to explore the use of CD98hc inhibitors as cancer therapeutics.

CD98hc (SLC3A2) regulation of skin homeostasis wanes with age.

Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal ß1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.

Role of the Notch ligand Delta1 in embryonic and adult mouse epidermis.

The Notch ligand Delta1 (Dll1) is expressed in human interfollicular epidermis (IFE) and regulates differentiation and adhesion of cultured human keratinocytes. However, the consequences of deleting Dll1 in mouse epidermis have not been examined. Here, we report that in embryonic mouse skin Dll1 is expressed by patches of keratinocytes in the basal layer of the IFE and in the dermal papilla and hair bulb. In a Dll1 hypomorph mutant that survives until birth, hair follicles formed normally but proliferation and thickness of the IFE were increased. Deletion of Dll1 using Cre recombinase expressed under the control of the keratin-5 (K5) promoter resulted in a delay in the first postnatal anagen, but subsequent hair cycles were normal. As in the hypomorph, IFE proliferation was stimulated and expression of K10 and K17 was disturbed. Older mice developed tumors with elements of IFE differentiation. Keratinocytes cultured from K5Cre x Dll1(flox/flox) epidermis showed a transient increase in proliferation, with a subsequent decrease in integrin expression and increased terminal differentiation. These results demonstrate that Dll1 contributes to the control of proliferation and differentiation in IFE, whereas Jagged1 regulates hair follicle differentiation.