Structure and Function of the Stratum Corneum Extracellular Matrix

Abstract

The stratum corneum (SC) extracellular matrix (ECM) is enriched in lipids that are organized into lamellar bilayers, whose molecular architecture is now known. Although these bilayers are important for the permeability barrier, the ECM contains not only lipids but also enzymes, structural proteins, and antimicrobial peptides that impact barrier function. Yet, how such diverse components affect barrier function remains largely unknown. Static models of the epidermis may not do justice to the ECM, which is metabolically active, as it changes both structure and function as it transits to the surface.

A brief history of our understanding of the stratum corneum interstices

Current understanding of the structure of stratum corneum (SC) began with the realization that its “normal basket-weave structure” in histologic sections is remarkably resilient, sharing characteristics with plastic wrap. Yet, the plastic wrap analogy is inaccurate; SC is not a homogenous sheet; rather, it is a geometrically arrayed, cellular tissue, with corneocytes arranged in vertical, interlocking columns (Christophers and Kligman, 1964). Frozen sections of SC, stained with lipid-detecting dyes, have shown that lipids in SC are segregated to membrane domains, a feature unique to the epidermis and other keratinizing epithelia, where they further localize to the extracellular matrix (ECM) of the SC, as shown by freeze-fracture replication and cell fractionation, which further demonstrates the organization of these lipids into lamellar bilayers (Elias et al., 1977). These multilayers form an expanded (≈10% of SC volume) domain that encloses corneocytes in a continuum of broad lamellar membranes (Elias and Friend, 1975).

Lamellar bilayer structure and function

Despite a lack of phospholipids, the approximately 1:1:1 molar ratio of ceramides (Cer), cholesterol, and nonessential fatty acids allows the incorporation of these lipids into unique multilamellar structures, with a characteristic substructure (e.g., White et al. (1988) and Bouwstra et al. (1991)). Several models have been proposed to illuminate how Cer, including certain Cer species that are further ω-esterified by linoleic acid (acylCer), are deployed within the lamellar bilayers. Although these models generally show cholesterol and free fatty acids lying alongside the N-acyl group of Cer, Iwai et al. (this issue, 2012) now propose that cholesterol, instead, partners with the sphingoid base of Cer and acylCer, with the N-acyl group then forming outward-extending arms.

As all three lipids are required to form the lamellar structures that regulate barrier function (Man et al., 1993), this new model could be validated by ascertaining whether pharmacologic blockade of cholesterol synthesis (Feingold et al., 1990) alters the predicted molecular organization within the lamellar bilayers. Similarly, blockade of either fatty acid and/or Cer synthesis could be used to further determine where each lipid moiety is deployed within the lamellar bilayers. Experiments of Nature, in the form of ichthyosis in inherited disorders of distal cholesterol metabolism (Elias et al., 2011), have been reported, in which cholesterol-deficient scales could be examined to address this issue. If this new model withstands such experimental verification, it would then prove useful in assessing how the lamellar bilayers contribute to the permeability barrier, while also predicting the subcellular routes by which hydrophobic and amphiphilic molecules might preferentially traverse the SC via the ECM.

The ECM contains more than lipids

The title of the paper by Iwai et al. (2012) is somewhat misleading, because SC membrane domains are heterogeneous (Figure 1), containing not only lamellar bilayers but also the following: (1) corneocyte envelopes; (2) the ω-hydroxyceramide monolayer that surrounds corneocytes (i.e., the corneocyte lipid envelope); and (3) several other secreted enzymatic and structural proteins generated from lamellar bodies. These include the cathelicidin antimicrobial peptide, LL-37, human β-defensin 2, and corneodesmosin, as well as several proteolytic enzymes and their respective inhibitors. Many of these constituents have been shown to contribute to permeability barrier function. For example, lamellar bilayers are disorganized in inherited disorders that modify the corneocyte envelope, such as transglutaminase 1–deficient lamellar ichthyosis and loricrin keratoderma (reviewed in Elias et al., 2010). Similarly, lamellar bilayer organization is altered when the corneocyte lipid envelope is lost, as in ALOX-deficient autosomal recessive ichthyosis, neutral lipid storage disease (Chanarin–Dorfman syndrome), and Refsum disease (Elias et al., 2010). Finally, transgenic ablation of the mouse cathelicidin protein results in a barrier abnormality owing to defective lamellar bilayer organization.

Figure 1.

Diagram of stratum corneum membrane domains (modified from Schmuth et al., 2008).

The larger question then becomes: how do all of these constituents participate in creating an effective, paracellular barrier to outward movement of water while preventing the ingress of noxious substances? Biophysical studies on the molecular architecture of the lamellar bilayers, although certainly important, have not yet taken into consideration the composite structural basis of the permeability barrier. Iwai et al. (2012) leave other questions unanswered: for example, they examined cryosections from an unspecified depth (mid-SC). Specifically, how does lamellar membrane architecture differ at varying levels of SC? These bilayers likely change in structure in response to the dynamic requirements of a highly cohesive structure that optimizes barrier function in the lower SC, which then relinquishes that role at more apical sites in preparation for shedding. Hence, information about the structure of lamellar bilayers deep in the mid-SC may not apply to bilayers at either more proximal or distal levels.

Where is the water?

There is also the issue of hydration; i.e., the site(s) at which water is sequestered in the SC. This much-debated subject is not trivial, as the SC’s ability to imbibe water allows humans to take baths and to swim in fresh water without drowning. Iwai et al. (2012) suggest that the lamellar bilayer architecture is not influenced by hydration, but the evidence for this claim is incomplete—a SC sample was immersed in water for 2 hours, after which no changes in membrane architecture appeared to take place. Yet, if the initial sample were already fully hydrated, one might anticipate no further change. To resolve this issue, future studies should include samples that are dried initially and then rehydrated. Similarly, the evidence that corneocytes alone imbibe water is based upon visual observations of the corneocyte interior alone. It should be noted that several (potentially water-binding) extracellular proteins are present in the ECM. Some are secreted by lamellar bodies, but others are derived from the degradation of corneodesmosomes. As corneodesmosomes are degraded, lacunae form, which then appear to provide an aqueous pore pathway through which hydrophilic molecules might traverse the ECM (Menon and Elias, 1997). In a report not yet available to the investigators (Lin et al., 2012), we have shown that these lacunar domains expand with hydration, as do the hydrophilic leaflets within the lamellar bilayers. Furthermore, the lamellar bilayers are degraded in the mid- to outer-SC following hydration, apparently through the activation of acidic ceramidases. It appeared that throughout these maneuvers only the extracellular compartment changed in volume with hydration. Thus, it would appear that the extracellular, rather than the cellular, compartment in the mid- to outer-SC expands and contracts in response to changes in hydration.

Limitations of biophysical approaches

In the final analysis, biophysical studies that assess the SC in a frozen state can provide valuable information about highly organized structures, such as intact lamellar bilayer arrays within the SC. As these approaches do not assess less-organized microdomains present within the ECM, there is potential for sampling bias in such studies. Yet, it is within these less-organized, highly plastic (dynamic) domains that subsequent investigators may find much of the “action”. Although Iwai et al. (2012) bring the field forward by presenting a more accurate model of the molecular organization of lamellar bilayers in the SC, it is too early to conclude that we understand the structural basis of the permeability barrier completely.

Clinical Implications.

• Molecular organization of the extracellular lamellar bilayers of the stratum corneum has now been refined, with implications for transcutaneous drug delivery and barrier function.

• The extracellular matrix of the stratum corneum contains more than lipids, and several of these nonlipid constituents are important for barrier function.

• The stratum corneum and its extracellular matrix are dynamic, changing in structure and function during transit to the surface of skin.