Skin fibroblast functional heterogeneity in health and disease.

Fibroblasts are the major cell population of connective tissue, including the skin dermis, and are best known for their function in depositing and remodelling the extracellular matrix. Besides their role in extracellular matrix homeostasis, fibroblasts have emerged as key players in many biological processes ranging from tissue immunity and wound healing to hair follicle development. Recent advances in single-cell RNA-sequencing technologies have revealed an astonishing transcriptional fibroblast heterogeneity in the skin and other organs. A key challenge in the field is to understand the functional relevance and significance of the identified new cell clusters in health and disease. Here, we discuss the functionally distinct fibroblast subtypes identified in skin homeostasis and repair and how they evolve in fibrotic disease conditions, in particular keloid scars and cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Fibroblast Heterogeneity in Healthy and Wounded Skin.

Fibroblasts are the main cell type in the dermis. They are responsible for the synthesis and deposition of structural proteins such as collagen and elastin, which are integrated into the extracellular matrix (ECM). Mouse and human studies using flow cytometry, cell culture, skin reconstitution, and lineage tracing experiments have shown the existence of different subpopulations of fibroblasts, including papillary fibroblasts, reticular fibroblasts, and fibroblasts comprising the dermal papilla at the base of the hair follicle. In recent years, the technological advances in single-cell sequencing have allowed researchers to study the repertoire of cells present in full-thickness skin including the dermis. Multiple groups have confirmed that distinct fibroblast populations can be identified in mouse and human dermis on the basis of differences in the transcriptional profile. Here, we discuss the current state of knowledge regarding dermal fibroblast heterogeneity in healthy mouse and human skin, highlighting the similarities and differences between mouse and human fibroblast subpopulations. We also discuss how fibroblast heterogeneity may provide insights into physiological wound healing and its dysfunction in pathological states such as hypertrophic and keloid scars.

Embigin is a fibronectin receptor that affects sebaceous gland differentiation and metabolism.

Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism.

Role of distinct fibroblast lineages and immune cells in dermal repair following UV radiation-induced tissue damage.

Solar ultraviolet radiation (UVR) is a major source of skin damage, resulting in inflammation, premature ageing, and cancer. While several UVR-induced changes, including extracellular matrix reorganisation and epidermal DNA damage, have been documented, the role of different fibroblast lineages and their communication with immune cells has not been explored. We show that acute and chronic UVR exposure led to selective loss of fibroblasts from the upper dermis in human and mouse skin. Lineage tracing and in vivo live imaging revealed that repair following acute UVR is predominantly mediated by papillary fibroblast proliferation and fibroblast reorganisation occurs with minimal migration. In contrast, chronic UVR exposure led to a permanent loss of papillary fibroblasts, with expansion of fibroblast membrane protrusions partially compensating for the reduction in cell number. Although UVR strongly activated Wnt signalling in skin, stimulation of fibroblast proliferation by epidermal ß-catenin stabilisation did not enhance papillary dermis repair. Acute UVR triggered an infiltrate of neutrophils and T cell subpopulations and increased pro-inflammatory prostaglandin signalling in skin. Depletion of CD4- and CD8-positive cells resulted in increased papillary fibroblast depletion, which correlated with an increase in DNA damage, pro-inflammatory prostaglandins, and reduction in fibroblast proliferation. Conversely, topical COX-2 inhibition prevented fibroblast depletion and neutrophil infiltration after UVR. We conclude that loss of papillary fibroblasts is primarily induced by a deregulated inflammatory response, with infiltrating T cells supporting fibroblast survival upon UVR-induced environmental stress.

Fibroblast Memory in Development, Homeostasis and Disease.

Fibroblasts are the major cell population in the connective tissue of most organs, where they are essential for their structural integrity. They are best known for their role in remodelling the extracellular matrix, however more recently they have been recognised as a functionally highly diverse cell population that constantly responds and adapts to their environment. Biological memory is the process of a sustained altered cellular state and functions in response to a transient or persistent environmental stimulus. While it is well established that fibroblasts retain a memory of their anatomical location, how other environmental stimuli influence fibroblast behaviour and function is less clear. The ability of fibroblasts to respond and memorise different environmental stimuli is essential for tissue development and homeostasis and may become dysregulated in chronic disease conditions such as fibrosis and cancer. Here we summarise the four emerging key areas of fibroblast adaptation: positional, mechanical, inflammatory, and metabolic memory and highlight the underlying mechanisms and their implications in tissue homeostasis and disease.

Distinct Fibroblast Lineages Give Rise to NG2+ Pericyte Populations in Mouse Skin Development and Repair.

We have examined the developmental origins of Ng2+ perivascular cell populations that adhere to the basement membrane of blood vessels, and their contribution to wound healing. Neural/glial antigen 2 (Ng2) labeled most perivascular cells (70-80%) in developing and adult mouse back skin, a higher proportion than expressed by other pericyte markers Tbx18, Nestin and Pdgfrß. In adult mouse back skin Ng2+ perivascular cells could be categorized into 4 populations based on whether they expressed Pdgfrα and Pdgfrß individually or in combination or were Pdgfr-negative. Lineage tracing demonstrated that although Ng2+ cells in embryonic and neonatal back skin contributed to multiple cell types they did not give rise to interfollicular fibroblasts within the dermis. Lineage tracing of distinct fibroblast populations during skin development showed that papillary fibroblasts (Lrig1+) gave rise to Ng2+ perivascular cells in the upper dermis, whilst Ng2+ perivascular cells in the lower dermis were primarily derived from reticular Dlk1+ fibroblasts. Following wounding of adult skin, Ng2+ dermal cells only give rise to Ng2+ blood vessel associated cells and did not contribute to other fibroblast lineages. The relative abundance of Ng2+ Pdgfrß+ perivascular populations was comparable in wounded and non-wounded skin, indicating that perivascular heterogeneity was maintained during full thickness skin repair. In the wound bed Ng2+ perivascular populations were primarily derived from Lrig1+ papillary or Dlk1+ reticular fibroblast lineages, according to the location of the regenerating blood vessels. We conclude that Ng2+ perivascular cells represent a heterogeneous lineage restricted population that is primarily recruited from the papillary or reticular fibroblast lineages during tissue regeneration.

Dissecting Fibroblast Heterogeneity in Health and Fibrotic Disease.

PURPOSE OF REVIEW: Fibroblasts, the major cell population in all connective tissues, are best known for their role in depositing and maintaining the extracellular matrix. Recently, numerous specialised functions have been discovered revealing unpredicted fibroblast heterogeneity. We will discuss this heterogeneity, from its origins in development to alterations in fibrotic disease conditions. RECENT FINDINGS: Advances in lineage tracing and single-cell transcriptional profiling techniques have revealed impressive diversity amongst fibroblasts in a range of organ systems including the skin, lung, kidney and heart. However, there are major challenges in assimilating the findings and understanding their functional significance. Certain fibroblast subsets can make specific contributions to healthy tissue functioning and to fibrotic disease processes; thus, therapeutic manipulation of particular subsets could be clinically beneficial. Here we propose that four key variables determine a fibroblast's phenotype underpinning their enormous heterogeneity: tissue status, regional features, microenvironment and cell state. We review these in different organ systems, highlighting the importance of understanding the divergent fibroblast properties and underlying mechanisms in tissue fibrosis.

The Roles of YAP/TAZ and the Hippo Pathway in Healthy and Diseased Skin.

Skin is the largest organ of the human body. Its architecture and physiological functions depend on diverse populations of epidermal cells and dermal fibroblasts. Reciprocal communication between the epidermis and dermis plays a key role in skin development, homeostasis and repair. While several stem cell populations have been identified in the epidermis with distinct locations and functions, there is additional heterogeneity within the mesenchymal cells of the dermis. Here, we discuss the current knowledge of how the Hippo pathway and its downstream effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) contribute to the maintenance, activation and coordination of the epidermal and dermal cell populations during development, homeostasis, wound healing and cancer.

Mutant Lef1 controls Gata6 in sebaceous gland development and cancer.

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a ß-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.

Fibroblast state switching orchestrates dermal maturation and wound healing.

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual-tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue-scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation.

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent monoamine oxidase that contributes to the remodelling of the extracellular matrix (ECM) by cross linkage of collagen and elastin fibres and has emerged as a potential therapeutic target in cancer and fibrosis. In the skin, LOXL2 is essential for epidermal cell polarity and differentiation. However, its role in the dermis has not been evaluated. We found that Loxl2 is dispensable for mouse dermal development, maturation and homeostasis, yet affects dermal stiffness. Neither loss of Loxl2 nor increased Loxl2 expression affected dermal architecture following treatment with the phorbol ester TPA. Furthermore, Loxl2 expression did not alter the stroma of DMBA-TPA-induced tumours. We conclude that, although Loxl2 is expressed in both dermis and epidermis, its function appears largely confined to the epidermis.

Skin Cell Heterogeneity in Development, Wound Healing, and Cancer.

Skin architecture and function depend on diverse populations of epidermal cells and dermal fibroblasts. Reciprocal communication between the epidermis and dermis plays a key role in skin development, homeostasis and repair. While several stem cell populations have been identified in the epidermis with distinct locations and functions, it is now recognised that there is additional heterogeneity within the mesenchymal cells of the dermis. Here, we discuss recent insights into how these distinct cell populations are maintained and coordinated during development, homeostasis, and wound healing. We highlight the importance of the local environment, or niche, in cellular plasticity. We also discuss new mechanisms that have been identified as influencing wound repair and cancer progression.

Dermal Blimp1 Acts Downstream of Epidermal TGFß and Wnt/ß-Catenin to Regulate Hair Follicle Formation and Growth.

B-lymphocyte-induced maturation protein 1 (Blimp1) is a transcriptional repressor that regulates cell growth and differentiation in multiple tissues, including skin. Although in the epidermis Blimp1 is important for keratinocyte and sebocyte differentiation, its role in dermal fibroblasts is unclear. Here we show that Blimp1 is dynamically regulated in dermal papilla cells during hair follicle (HF) morphogenesis and the postnatal hair cycle, preceding dermal Wnt/ß-catenin activation. Blimp1 ablation in E12.5 mouse dermal fibroblasts delayed HF morphogenesis and growth and prevented new HF formation after wounding. By combining targeted quantitative PCR screens with bioinformatic analysis and experimental validation we demonstrated that Blimp1 is both a target and a mediator of key dermal papilla inductive signaling pathways including transforming growth factor-ß and Wnt/ß-catenin. Epidermal overexpression of stabilized ß-catenin was able to override the HF defects in Blimp1 mutant mice, underlining the close reciprocal relationship between the dermal papilla and adjacent HF epithelial cells. Overall, our study reveals the functional role of Blimp1 in promoting the dermal papilla inductive signaling cascade that initiates HF growth.

Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells.

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.

Inhibition of ß-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing.

New hair follicles (HFs) do not form in adult mammalian skin unless epidermal Wnt signalling is activated genetically or within large wounds. To understand the postnatal loss of hair forming ability we monitored HF formation at small circular (2 mm) wound sites. At P2, new HFs formed in back skin, but HF formation was markedly decreased by P21. Neonatal tail also formed wound-associated HFs, albeit in smaller numbers. Postnatal loss of HF neogenesis did not correlate with wound closure rate but with a reduction in Lrig1-positive papillary fibroblasts in wounds. Comparative gene expression profiling of back and tail dermis at P1 and dorsal fibroblasts at P2 and P50 showed a correlation between loss of HF formation and decreased expression of genes associated with proliferation and Wnt/ß-catenin activity. Between P2 and P50, fibroblast density declined throughout the dermis and clones of fibroblasts became more dispersed. This correlated with a decline in fibroblasts expressing a TOPGFP reporter of Wnt activation. Surprisingly, between P2 and P50 there was no difference in fibroblast proliferation at the wound site but Wnt signalling was highly upregulated in healing dermis of P21 compared with P2 mice. Postnatal ß-catenin ablation in fibroblasts promoted HF regeneration in neonatal and adult mouse wounds, whereas ß-catenin activation reduced HF regeneration in neonatal wounds. Our data support a model whereby postnatal loss of hair forming ability in wounds reflects elevated dermal Wnt/ß-catenin activation in the wound bed, increasing the abundance of fibroblasts that are unable to induce HF formation.

Integrins synergise to induce expression of the MRTF-A-SRF target gene ISG15 for promoting cancer cell invasion.

Integrin-mediated activation of small GTPases induces the polymerisation of G-actin into various actin structures and the release of the transcriptional co-activator MRTF from G-actin. Here we report that pan-integrin-null fibroblasts seeded on fibronectin and expressing ß1- and/or αV-class integrin contained different G-actin pools, nuclear MRTF-A (also known as MKL1 or MAL) levels and MRTF-A-SRF activities. The nuclear MRTF-A levels and activities were highest in cells expressing both integrin classes, lower in cells expressing ß1 integrins and lowest in cells expressing the αV integrins. Quantitative proteomics and transcriptomics analyses linked the differential MRTF-A activities to the expression of the ubiquitin-like modifier interferon-stimulated gene 15 (ISG15), which is known to modify focal adhesion and cytoskeletal proteins. The malignant breast cancer cell line MDA-MB-231 expressed high levels of ß1 integrins, ISG15 and ISGylated proteins, which promoted invasive properties, whereas non-invasive MDA-MB-468 and MCF-7 cell lines expressed low levels of ß1 integrins, ISG15 and ISGylated proteins. Our findings suggest that integrin-adhesion-induced MRTF-A-SRF activation and ISG15 expression constitute a newly discovered signalling circuit that promotes cell migration and invasion.