The stratum corneum barrier: impaired function in relation to associated lipids and proteins

ABSTRACT

The skin is the largest organ of the human body and is widely considered to be the first-line defense of the body, providing essential protection against mechanical, physical, and chemical damage. Keratinocytes are the primary cells of the outer layer of the epidermis, which acts as a mechanical and permeability barrier. The epidermis is a permanently renewed tissue where undifferentiated keratinocytes located at the basal layer proliferate and migrate to the overlying layers. Here we report that some components of keratinocytes affect the formation and differentiation of the stratum corneum, which is the most specialized layer of the epidermis.

Introduction

The skin is a barrier that separates the host from the external environment with physical, chemical, microbial, and immune components.^1,2 The epidermis, dermis, and subcutaneous layer make up the skin: the epidermis is a thin layer of stratified squamous epithelium composed of four layers of keratinocytes with different degrees of differentiation, providing a waterproof barrier to the external environment and preventing excessive water loss from the body;³ the dermis, located below the epidermis, is a structure composed of the extracellular matrix and skin appendages such as pilosebaceous units, blood vessels, lymphatics. The deepest layer is the subcutaneous layer, composed of subcutaneous fat and connective tissue.⁴ As the external layer of the skin, the barrier function of the epidermis protects the host from the constant challenge of pathogens in the environment,¹ which can be divided into the stratum corneum barrier, microbial barrier, sebum barrier, and immune barrier. In the stratum corneum, keratinocytes become morphologically flattened and lose their nuclei, and the intercellular spaces are filled with lipids.⁵ Together, they provide a highly hydrophobic barrier to the environment. Thus, the barrier function of the stratum corneum is further enhanced and is the most unique structure in the epidermis. The outermost layer of the epidermis is colonized by complex and diverse bacteria and fungi that live in symbiosis with the organism and provide direct defense against pathogenic microorganisms.⁶ The sebum film is a liquid film formed on the skin surface by the emulsification of oil secreted from sebaceous glands, lipids produced by keratinocytes, sweat secreted from sweat glands, and shed keratinocytes at low temperatures, which has the function of softening the skin, regulating the water content of the epidermis, and preventing external organisms.^7–9 In the epidermis, immune cells such as Langerhans cells, γδ T cells, and CD8 resident memory T cells are located between keratinocytes to maintain homeostasis when the epidermis is damaged.¹ Stratum corneum, a surface barrier of the epidermis, is formed by the continuous proliferation, terminal differentiation, and pushing up of keratinocytes located in the basal layer at the base of the epidermis.¹⁰

The barriers of the epidermis are interconnected with the stratum corneum barrier as the core (Figure 1). Epidermal commensal microbiome influences host metabolism during pathology and trauma in the medium of keratinocytes,¹¹ while the microbiota is associated with the development of many autoimmune diseases,¹² which are directly related to the disruption of the stratum corneum leading to the entry of pathogens into the epidermis.¹³ Lipids are secreted by the sebaceous glands and pass through the stratum corneum to cover the surface and protect it from potential toxins,¹⁴ sebum provides skin microbiota with the substrates required for lipid metabolism,¹⁵ and the sebum barrier further inhibits the growth of deleterious bacteria. For the immune barrier, the stratum corneum barrier is well insulated from immune irritants, and conversely, damage to the stratum corneum can likewise cause an immune response, known as skin sensitivity or allergy.¹⁶ Overall, the synergy of these multiple layers of barriers constitutes the complete skin barrier function.

Figure 1.

Relationship between multiple epidermal barriers. the epidermal barrier is centrally interconnected with the stratum corneum barrier. The epidermal commensal microbiota colonizes the outer stratum corneum and influences host metabolism between pathologies, and the microbiota is associated with the development of many autoimmune diseases. The lipids that make up the sebum barrier are secreted by the sebaceous glands and protect the stratum corneum from external aggressions, sebum provides the substrate required for lipid metabolism by the skin microbiota, and the sebum barrier further inhibits the growth of harmful bacteria. The stratum corneum barrier isolates immune stimulants, and damage to the stratum corneum causes an immune response.

Accumulating evidence demonstrates that damage to the stratum corneum is one of the key factors of skin sensitivity and allergic disorders.^5,17 Although sensitive skin syndrome is a subjective sensation, it is widely recognized that skin barrier dysfunction is associated with sensitivity as well as, critically, impaired functions of the stratum corneum barrier, in particular, increased transcutaneous penetration of water-soluble chemicals due to stratum corneum thinning and intercellular lipid imbalance are important mechanisms leading to sensitive skin.^18,19

Over the years, numerous studies have shown skin components have a profound effect on the stratum corneum. In this review, we will first discuss how structural proteins and intercellular lipids like filaggrin (FLG), keratins, and ceramides are involved in regulating stratum corneum function, and secondly, the influence of immune factors on the differentiation process of keratinocytes that form the stratum corneum.

Filaggrin maintains the mechanical strength of keratinocytes in the stratum corneum

Filaggrin is processed from profilaggrin, which is produced by differentiated keratinocytes in the stratum granulosum and deposited in the keratohyalin granules.²⁰ Concretely, during the late stages of the keratinocyte terminal differentiation, profilaggrin is secreted into the cytoplasm, where it is rapidly dephosphorylated and proteolyzed into filaggrin through multiple proteolytic steps.^20–22 Filaggrin is a major structural protein of the stratum corneum, which binds to keratin intermediate filaments to form the dense keratin matrix that facilitates the mechanical strength and integrity of the stratum corneum and the skin barrier function^23–26 (Figure 2). In addition, filaggrin is further completely decomposed to hygroscopic amino acids by peptidylarginine deiminases²⁷ and their derivatives by proteases in the stratum corneum for tissue hydration^28–30 (Figure 2). It is worth mentioning that recent research has discovered filaggrin, the primary constituent of the cornified envelope, exists as a liquid-like condensate within cells, exhibiting mechanical responsiveness. As granular cells migrate toward the epidermis and undergo terminal differentiation, filaggrin facilitates nuclear degradation, promoting the pH-dependent release of proteases and nucleases from cornified envelope granules. This, in turn, facilitates the self-destruction phase of terminal differentiation.³¹ Inadequate expression of filaggrin or mutations triggers abnormal dry skin and even skin diseases^32,33 such as ichthyosis vulgaris,³⁴ food allergy, eosinophilic esophagitis,³⁵ and even asthma.³⁶ When the filaggrin gene was knocked out in mice, Flg^−/− mice exhibited dry scaly skin without concomitant impairment of stratum corneum hydration.³⁷ However, low-vacuum scanning electron microscopy and transmission electron microscopic analyses revealed that the deficiency of filaggrin resulted in the disabling loss of the keratin framework interspersed in 3-dimensions with a hexagonal pattern, leading to easier detachment of the stratum corneum under mechanical stress.³⁷ Moreover, filaggrins, which contain a large number of histidine residues, are a major source of histidine. Histidine has an important role in preventing UVB from penetrating the stratum corneum through the generation of urocanic acid by histidase. After the knockdown of filaggrin, DNA damage, and apoptosis were increased in UVB-exposed organotypic skin cultures, suggesting that filaggrin and their metabolism are associated with the UV sensitivity of stratum corneum.³⁸

Figure 2.

FLG production process, factors regulating FLG expression, and FLG-induced lipid production. i. Profilaggrin is secreted into the cytoplasm by keratohyalin granules to break down into filaggrin. ii. It binds to keratin intermediate filaments to form a dense keratin matrix, which facilitates the mechanical strength and integrity of the stratum corneum and the skin barrier function. iii. Filaggrin monomers are further decomposed to hygroscopic amino acids and their derivatives in the stratum corneum by proteases for skin hydration. Lysophosphatidic acid (LPA) binding to LPAR1/5 activates the RHO-ROCK-SRF pathway via G12/13, leading to the activation of the transcription factor SRF, which induces FLG gene expression and protein production. Filaggrin acts to inhibit α-toxin pathogenicity by mediating the secretion of SMPD to reduce the number of α-toxin binding sites on the surface of keratinocytes.

Despite these findings, FLG is essential for maintaining stratum corneum integrity, the intrinsic factors that regulate FLG expression and detailed mechanisms remain elusive³² (Figure 2). The lysophosphatidic acid receptor 1 (LPAR1) antagonist AM095 and the LPAR5 antagonist TCLPA54 significantly inhibit LPA-induced FLG expression in normal human epidermal keratinocytes (NHEKs).³² LPAR1/5 have been proven to activate the RHO-ROCK-SRF pathway via G_12/13, leading to the activation of transcription factor SRF in NHEKs, which induces FLG gene expression and protein production.³² Additionally, the expression of FLG promotes the proliferation of cornification-forming cells in 3D human epidermal culture systems, leading to an increase in the thickness of the stratum corneum.^39,40 Similarly, LPA promotes profilaggrin and filaggrin production and keratinocyte differentiation in the stratum granulosum and stratum corneum via the LPAR1/5-RHO-ROCK pathway under such conditions.³²

In addition to being involved in regulating keratinocyte mechanical strength, FLG may protect keratinocytes by regulating biological signaling (Figure 2). α-toxin, a tissue-damaging cytolysin, is produced by Staphylococcus aureus, rather contributes to the development and the exacerbation of atopic dermatitis.^41,42 Sphingomyelinase (SMPD) enzymatic decomposes sphingomyelin, a major component of the cell membrane, reducing the number of α-toxin binding sites on the surface of keratinocytes, thereby preventing toxin binding to cells, and FLG acts to inhibit the pathogenicity of α-toxin by mediating SMPD secretion.⁴³ Reduced FLG expression in the skin of atopic dermatitis (AD) patients could also indicate that FLG is involved in protecting keratinocytes in AD.⁴³

Keratin forms a strong barrier to the stratum corneum

Intermediate filaments (IFs) and microfilaments, along with microtubules, constitute essential components of the epidermal cytoskeleton. Keratin, as the largest subgroup of IF proteins, serves as a scaffold that enables cells to stand up to physical stress. Traditionally, this perspective has solely focused on the structural scaffolding role of keratins. However, in recent years, an increasing number of studies have unveiled additional functions of keratins and their intricate associations with tight junctions (TJs)⁴⁴ and other functions (Table 1).

Table 1.

Regulation of skin by keratin.

Keratin	Function	Experiment subject	Relate signal/protein expressed in the deficient animal model	Ref
Type 1 keratin cluster	Maintain shape	Keratinocyte cell sheets	Junctional actin and fibers actomyosin contractility decrease; total protein levels of adherens junction protein, occludin, claudin-1, and −4 absent	[45]
Keratin 1	Activate inflammation	Krt1−/− mice	Enhance expression of antimicrobial peptides S100A8 and S100A9; upregulate IL-18	[46]
Keratin 16	Activate inflammation	Krt16± and Krt16−/− mice	Hyper-activate DAMPs expression; overexpress IL-1β	[47]
Keratin 17	Encode AMP and lipid synthesis-related genes	Krt17−/− mice	Promotes the nuclear localization of SREBP-1	[48]
Keratin 76	Heal wounds	Krt76tm1a mice	Hyper-proliferation and induction of damaged keratins; mislocalization of membranous CLDN1	[44]

Keratinocytes lacking the entire type I keratin cluster (KtyI^−/−) have difficulty maintaining shape after mechanical disruption and impair mechanical coupling. This alteration was attributed to impaired junctional integrity of keratin-deficient cells, including a significant reduction in junctional actin fibers and a decrease in overall actomyosin contractility. In addition, the actin-linked adherents and tight junctions were also severely affected: total protein levels of adherens junction protein were reduced and occludin, claudin-1, and −4 were almost completely absent, indicating that cell-cell borders formation was disturbed in KtyI^−/− cells. Even though the re-expression of K5/K14 restores partial barrier function, relative keratin abundance remains a key factor in cellular mechanical properties and tight junction function.⁴⁵

Krt76-deficient mice exhibit overall epidermal thickening and fail to heal wounds spontaneously with age.⁴⁴ Immunofluorescence staining showed that normal mice exhibit Krt76 protein upregulation in healing wounds after injury in response to skin injury and to promote wound healing.⁴⁴ Krt76 mutant mice developed phenotypes associated with barrier dysfunction such as hyper-proliferation and abnormal differentiation processes, as indicated by overexpression of a basal keratin marker (KRT14), a marker of the stratum spinosum (KRT10), and filaggrin, which appears to be related to tight junctions, primarily a mislocalization of membranous CLDN1.⁴⁴ Immunoprecipitation experiments showed co-localization between CLDN1 and KRT76, which is important for mediating barrier dysfunction and wound healing phenotypes.⁴⁴

Keratin also plays a role in inflammatory signaling in response to cellular injury. When the epidermal barrier is compromised, keratinocytes produce the damage-associated molecular patterns (DAMPs) that activate immune cells and regulate cytokines to initiate the inflammatory response.⁴ Meanwhile, stressed keratinocytes rapidly induce the transcription of keratins that play a special role in inflammation and wound healing. However, keratinocytes lacking Krt16 hyper-activate DAMPs expression and overexpress alarm proteins such as IL-1β, causing acute skin inflammation.⁴⁷ Krt1^−/− mice upregulate IL-18 and enhance the expression of antimicrobial peptides S100A8 and S100A9 (DAMP associated with impaired skin barrier function and inflammatory skin diseases). In addition, NOD-like receptors (NLRs) activation of inflammasomes and caspase-1 in stressed keratinocytes cleaves IL-18 into its active form and releases it.⁴⁶

Keratin 17 is induced to be expressed after epidermal barrier disruption and is involved in epidermal permeability barrier recovery by regulating lipid metabolism. The expression of genes encoding antimicrobial peptides (S100A8, S100A9, LL-37) and proteins involved in lipid synthesis (fatty acid synthase and PPAR-gamma) are markedly reduced in Krt17^−/− mice after barrier disruption, and experiments using HaCat keratinocytes showed the effect of KRT17 silencing on sterol regulatory element-binding protein 1 (SREBP-1) localization, which is a transcription factor of lipid metabolism-related enzymes, positively regulating fatty acid synthase and peroxisome proliferator-activated receptor γ (PPARγ) expression in keratinocytes.⁴⁸

Ceramide synthesis process is essential to the permeation barrier

The epidermal permeability barrier consists mainly of keratinocytes and extracellular lipids in the stratum corneum, forming a “brick-mortar” structure. In addition to providing precursors to form the cornified lipid envelope of corneocyte, participate in the arrangement of extracellular lipids into specialized structures called lamellae, another lipid structure essential for the epidermal permeability barrier. These hydrophobic lipid matrices are protective against water and electrolyte loss. These lipids are primarily synthesized by lamellar bodies (LBs) in the upper stratum spinosum and stratum granulosum, an organelle with lipid storage and secretion functions, containing lipids, hydrolases, and other contents. Epidermal ceramides (Cers) are synthesized in the endoplasmic reticulum (ER) and subsequently translocated to the Golgi apparatus, where the glucose residues of UDP-glucose are transferred to ceramide by UDP-glucose ceramide glucosyltransferase (UGCG) to produce glucosylceramides (GlcCers),⁴⁹ which is the main component of LBs and the precursor of Cers⁵⁰ (Figure 3). After the fusion of LB with the apical plasma membrane of the stratum granulosum, LB exocytoses lipids and enzymes into the extracellular spaces of the stratum corneum to complete the final processing step of lipid precursors. Through the action of key enzymes such as β-glucocerebrosidase and ceramidase, GlcCer precursors are converted into Cers, fatty acids (mainly linoleic acid), and then form corneocyte lipid envelopes, which are covalently bound to the surface of keratinocyte envelopes composed of proteins to establish a skin permeation barrier.⁵¹ Ceramide deficiency leads to dry skin, which is why it is a key ingredient in many skin repair products.⁵² For example, Kim and colleagues have designed and constructed carriers with this multilayered ceramide core-structure to restore skin barrier function.⁵³

Figure 3.

Main pathways of ceramide synthesis. in the ER, serine and palmitoyl-CoA are condensed to 3-ketosphinganine (3-ketoSph) by serine palmitoyltransferase (SPT); 3-ketodihydrosphingosine reductase (KDSR) reduces it to D-sphingosine; D-sphingosine is produced as dihydroceramide (dhCer) by ceramide synthase (CERS); Dihydroceramide is desaturated by dihydroceramide desaturase (DES) to generate ceramide (Cer). In the Golgi, Cer is produced as glucose ceramides (GlcCer) via UDP-glucose ceramide glucosyltransferase (UGCG), and GlcCer are converted to Cer by β-glucocerebrosidase (β-GBA) and ceramidase. In the plasma membrane, sphingomyelin (SM) are decomposed by sphingomyelinase (SMPD) to ceramides.

Undoubtedly, Cer-glucosylation is the critical step in Cers processing. A decrease in GlcCer and protein-bound Cers was observed in the skin of UGCG-deficient mice and resulted in delayed keratinocyte differentiation, aberration of the junction proteins, and breakdown of the permeability barrier, manifested by epidermal water loss as well as desquamation.⁵⁴ In addition to the lipids secreted by the LBs, the lipids on the skin surface are derived from the sebaceous gland (SG). Sebum, which is composed of triglycerides (TG), wax esters (WE), and squalene, secreted by the SG is influenced by its distribution.⁵⁵

Most of the 16 ceramide isoforms in the stratum corneum consist of variable chain fatty acids as well as a sphingosine base, based on which ceramides can be categorized and 4-letter nomenclature.⁵⁶ The first 1–2 letters represent the acyl chain, including N (non-hydroxy), A (α-hydroxy), O (ω-hydroxy), and EO (esterfied-ω-hydroxy); the last 1–2 letters represent the sphingosine bases, including S (sphingosine), dS (sphinganine), P (phytosphingosine) and H (6 hydroxy sphingosine), see Table 2.⁵⁷

Table 2.

Stratum corneum ceramide subclasses and nomenclature.

	S (sphingosine)	dS (sphinganine)	P (phytosphingosine)	H (6-hydroxy sphingosine)
N (non-hydroxy)	NS	NdS	NP	NH
A (α-hydroxy)	AS	AdS	AP	AH
O(ω-hydroxy)	OS	OdS	OP	OH
EO(esterfied-ω-hydroxy)	EOS	EdS	EP	EH

Cytokines regulate keratinocyte differentiation

Keratinocytes proliferate and differentiate from the basal layer and eventually migrate to the stratum corneum to form terminally differentiated keratinocytes. The differentiation of keratinocytes is regulated by several factors (Figure 4). An increase in the number of mast cells and elevated levels of their released signaling molecule histamine have been observed in the skin of patients with atopic dermatitis.^58,59 Exogenous histamine reduces stratum corneum thickness in organotypic skin models and blocks the initiation of late differentiation programs in keratinocytes through the activation of histamine receptor-1, such as inhibition of late markers like filaggrin, loricrin, and keratin 10.⁶⁰ Simultaneously, epidermal tight junction proteins (ZO-1, Occludin, Claudin-1, Claudin-4) and desmosomal junction (corneodesmosin and desmoglein-1) showed reduced expression after histamine treatment, demonstrating skin barrier dysfunction.⁶⁰

Figure 4.

Immunomodulatory factors in keratinocyte differentiation. Th2 cytokines IL-4 and IL-13 affect structural components and barrier function at early stages of keratinocyte differentiation; exogenous histamine prevents the initiation of late keratinocyte differentiation program through activation of histamine receptor-1; AKT1 is expressed at late stages of terminal differentiation and AKT1 may affect barrier function in the keratinocyte layer by regulating nuclear degradation.

T helper type 2 (Th2)-immune response is involved in the development of allergic diseases,⁶¹ while Th2 cytokines IL-4 and IL-13 influence structural components and barrier function at the early stages of keratinocyte differentiation. In the early stages of human and mouse keratinocyte differentiation, IL-4 and IL-13 disrupt the integrity of cell sheets by downregulating the mRNA expression levels of Krt1, Krt10, Dsg1a, and Dsc1 through IL-4 Rα and increasing the sensitivity of the keratinocyte cell sheets to mechanical stress.⁶² In particular, STAT6 and p44/42 MAPK signaling pathways mediate the downregulation of Krt1 and Krt10 expression by IL-4 in mouse keratinocytes, whether this is the same as the signaling pathway in human keratinocytes remains to be explored.⁶²

The stratum corneum is renewed by the integration of dead cells, and thus cellular remodeling during cornification leads to a protective skin barrier. Since pro-inflammatory signals from dying cells activate skin inflammation, the process of cornification, especially the terminal stage, is closely related to inflammation, which makes cornification a protective mechanism with an inhibitory effect on inflammation particularly important.⁶³ Parakeratosis, commonly observed in many epidermal barrier-deficient diseases⁶⁴ and during wound healing,⁶⁵ is a phenomenon in which nuclear material is retained in the stratum corneum. As an essential signaling molecule for epidermal terminal differentiation, researchers hypothesize that AKT1 may affect the barrier function of the stratum corneum by regulating nuclear degradation. AKT1 is expressed during late terminal differentiation and phosphorylates Lamin A/C to degrade it in keratinocytes.⁶⁶ Lamin A/C is a scaffold for the nucleus, and degradation causes the nuclear lamina to rupture to allow DNAase to enter the nucleus to complete the nuclear degradation process for the terminal differentiation of keratinocytes.⁶⁷ AKT1 not only regulates nuclear degradation but also affects keratinocyte differentiation. AKT1 defects prevent nuclear material spillover by blocking Lamin A/C degradation, thus allowing nuclear material retained in the keratinocyte layer to reduce levels of differentiation markers keratin 1, keratin 10, filaggrin, and Loricrin through the BMP2/SMAD1 pathway,⁶⁶ causing paracrine changes to keratin and loricrin expression. However, BMP2 is reported to be present exist outside the cell,⁶⁸ and how the undisclosed nuclear material activates BMP2 in the nucleus has not been clearly investigated.

Conclusion

This review discussed the intricate interplay of skin components affecting the stratum corneum, focusing on FLG, keratin, and lipids. Additionally, it elucidates the impact of histamine in atopic dermatitis and Th2 cytokines in allergic diseases on the proteins involved in keratinocyte differentiation. Despite the progress made, numerous questions linger unanswered. For instance, the involvement of keratin 17 in encoding antimicrobial peptides and lipid synthesis, as well as the alterations in keratinocyte differentiation dynamics during wound healing, warrant further exploration. The skin barrier, with the stratum corneum as its cornerstone, plays a pivotal role in skin sensitivity (Figure 5). We anticipate that unraveling the intricate mechanisms underlying the interaction between the stratum corneum and skin sensitization will enable the development of more effective strategies to target and regulate the stratum corneum barrier. Moreover, the integration of cutting-edge techniques such as single-cell RNA sequencing and spatial transcriptomics holds promise in shedding light on the complex orchestration of epidermal functions. These advanced methodologies offer unprecedented opportunities to decipher the molecular signatures and spatial organization underlying skin barrier function, paving the way for innovative therapeutic interventions in dermatological conditions.

Figure 5.

Skin barrier changes during skin sensitization. Skin sensitization manifests as dry, flaky, painful and reddened skin. Dryness and increased permeability are caused by thinning of the sebum and stratum corneum; subsequent activation of inflammation causes redness and pain; and sensitive skin is often accompanied by a decrease in the diversity of the microbiome.

Abbreviations

FLG

Filaggrin

LPAR1

Lysophosphatidic acid receptor 1

NHEKs

Normal human epidermal keratinocytes

SMPD

Sphingomyelinase

AD

Atopic dermatitis

IFs

Intermediate filaments

TJs

Tight junctions

DAMPs

Damage-associated molecular patterns

NLRs

NOD-like receptors

SREBP-1

Sterol regulatory element-binding protein 1

PPARγ

Peroxisome proliferator-activated receptor γ

LBs

Lamellar bodies

Cers)

Ceramides

UGCG

UDP-glucose ceramide glucosyltransferase

GlcCers

Glucosylceramides

SG

Sebaceous gland

TG

Triglycerides

WE

Wax esters

Th2

T helper type 2

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions statement

H Qian and X Gong participated in the conception and design of the review. J Chen and Y Yang participated in literature research and drew the figures. J Chen, CJ Liu and X Gong wrote the manuscript.