In fully cornified cells, filaggrin is degraded into free amino acids. This high concentration of hydrophillic amino acids is essential for water retention and contributes to the osmolarity, and consequently the flexibility of the cornified layer (Candi et al. 2005). Filaggrin monomers are a direct target for cleavage by the aspartate-specific protease caspase 14 (Denecker et al. 2007, 2008, Hoste et al. 2011, Eckhart & Tschachler 2011). Proteases able to process profilaggrin into fillagrin in vitro include microbial ST14 (Profillagrin endopeptidase 1, PEP1), CAPN1 (mu-calpain), furin, PACE4 and matriptase MT-SP1 (Reising et al. 1995, Yamazaki et al. 1997, Pearton et al. 2001, List et al. 2003, Candi et al. 2005), but these proteases do not appear to have a role in the degradation of filaggrin that occurs at a late stage in keratinization.

Filaggrin is initially synthesized as a large, insoluble, highly phosphorylated precursor containing many tandem copies of 324 residues. This precursor is dephosphorylated and proteolytically cleaved by several proteases, including the undefined protease PEP1 (Resing et al. 1996), mu-calpain (Yamazaki et al. 1997), furin, PCSK6 (PACE4) (Pearton et al. 2001), PRSS8 (cap1) (Leyvraz et al. 2005), ST14 (matriptase) (List et al. 2003), CELA2 (Bonnart et al. 2010), CASP14 (Denecker et al. 2007) and Kallikrein-related peptidase 5 (KLK5) (Sakabe et al. 2013). Filaggrin is further processed and proteolytically degraded by CASP14 (Eckhart & Tschachler 2011).

Filaggrin is initially synthesized as a large, insoluble, highly phosphorylated precursor containing many tandem copies of 324 residues. This precursor is dephosphorylated and proteolytically cleaved by several proteases, including the undefined protease PEP1 (Resing et al. 1996), mu-calpain (Yamazaki et al. 1997), furin, PCSK6 (PACE4) (Pearton et al. 2001), PRSS8 (cap1) (Leyvraz et al. 2005), ST14 (matriptase) (List et al. 2003), CELA2 (Bonnart et al. 2010), CASP14 (Denecker et al. 2007) and Kallikrein-related peptidase 5 (KLK5) (Sakabe et al. 2013). Filaggrin is further processed and proteolytically degraded by CASP14 (Eckhart & Tschachler 2011).

During differentiation of stratum granulosum keratinocytes into corneocytes, lytic enzymes are activated by the influx of Ca2+ and all metabolic activity ceases. Corneocytes, in the form of flattened squames, represent cellular skeletons filled with keratin filaments and sealed together by lipids. Corneocytes form an impermeable and insoluble barrier that protects against the invasion of microorganisms and loss of bodily fluids (reviewed by Fuchs 1990).

Markers of granular keratinocytes are summarized in the "Table of granular keratinocytes in interfollicular epidermis". Some of the markers, in particular AQP3, LGLAS7, and FABP5, are described in more detail in the sections "Keratinocyte stem cell differentiates into transit amplifying cell in the basal layer of interfollicular epidermis" and "Keratinocyte of spinosum layer differentiates into keratinocyte of granulosum layer in interfollicular epidermis".

CALML5 (Calmodulin-like protein 5, also known as Calmodulin-like skin protein or CLSP) is expressed in keratinocytes of stratum granulosum and lower layers of stratum corneum and is a marker of late keratinocyte differentiation (Mehul et al. 2001). CALML5 is proteolytically degraded in upper layers of normal but not psoriatic stratum corneum, which is prevented by Ca2+ (Mehul et al. 2006). CALML5 is implicated in regulation of function of Ca2+-dependent proteins involved in skin barrier formation (Donovan et al. 2015). By RNA sequencing, CALML5 is the most enriched gene in differentiating outer epidermis. CALML5 gene transcription is upregulated by ZNF750 transcription factor and CALML5 mRNA is stabilized by noncoding RNA TINCR; CALML5 knockdown interferes with terminal differentiation of keratinocytes, formation of keratohyalin granules, and the skin barrier function. SFN is a binding partner of CALML5 (Sun et al. 2015).


Table of markers of granular keratinocytes in interfollicular epidermis. Please note that keratinocytes in CellMarker database and PanglaoDB correspond to granular keratinocytes in Reactome.

Marker (protein/RNA)	Literature Reference	CellMarker database – RNA/Protein (Hu et al. 2022)	PanglaoDB – RNA (Franzén et al. 2019)
AQP3 (protein)	Sugiyama et al. 2001 Ma et al. 2002 Sougrat et al. 2002	No	Yes
CALML5 (protein)	Mehul et al. 2001 Mehul et al. 2006 Donovan et al. 2015 Sun et al. 2015	No	Yes
DMKN (protein)	Matsui et al. 2004 Higashi et al. 2012 Leclerc et al. 2014	No	Yes
FAPB5 (protein)	Let et al. 1998	No
FLG (protein)	Smoller et al. 1990 Steven et al. 1990 Juhlin et al. 1992 Girbal Neuhauser et al. 1997 Yoneda et al. 2012 Sakabe et al. 2013 Wong et al. 2015 reviewed in McLean 2016 reviewed in Ishida Yamamoto et al. 2018	No	Yes
GJB3 (protein)	Butterweck et al. 1994 Di et al. 2001	No	Yes
HOPX (protein)	Obarzanek Fojt et al. 2011	No	No
IVL (protein)	Bernard et al. 1985 Smoller et al. 1990 van Duijnhoven et al. 1992 Ishida Yamamoto et al. 1996 Crish et al. 2002 Chen et al. 2013	No	Yes>
KLK5 (protein)	Ishida Yamamoto et al. 2005 Sakabe et al. 2013 Miyai et al. 2014	No	No
KLK7 (protein)	Ishida Yamamoto et al. 2005 Miyai et al. 2014	No	No
KPRP (protein)	Lee et al. 2005 Suga et al. 2019 Jeriha et al. 2020	No	Yes
KRT1 (protein)	reviewed in Fuchs 1990 Totsuka et al. 2017	No	Yes
KRT10 (protein)	Yoneda et al. 2012 Totsuka et al. 2017	No	Yes
KRTDAP (protein)	Matsui et al. 2004 Tsuchida et al. 2004	No	No
LGALS7 (protein)	Magnaldo et al. 1995 Umayahara et al. 2020	No	Yes
LORICRIN (protein, RNA)	Steven et al. 1990 Juhlin et al. 1992 Magnaldo et al. 1992 Ishida Yamamoto et al. 1996 reviewed in Ishida Yamamoto et al. 1998 Yoneda et al. 2012 reviewed in Ishida Yamamoto et al. 2018	No	No
PRSS3 (protein)	Nakanishi et al. 2010 Miyai et al. 2014	No	No
SBSN (protein)	Matsui et al. 2004 Bazzi et al. 2007 Aoshima et al. 2019	No	Yes
SPINK5, also known as LEKTI (protein)	Bitoun et al. 2003 Ishida Yamamoto et al. 2005 Galliano et al. 2005 reviewed in Ishida Yamamoto et al. 2018	No	No
SPRR2A (protein)	Hohl et al. 1995 Sark et al. 1998 Cabral et al. 2003	Yes	No

DMKN (dermokine)-endcoding gene is part of the keratinocyte related locus on human chromosome 19q13.1 that also includes SBSN and KRTDAP genes. Coordinated transcription of these genes is detectable in stratum spinosum, but proteins are the most prominent in the granular layer keratinocytes, from which they are secreted. DMKN has several splicing isoforms, of which alpha, beta, and gamma are known to be expressed in the epidermis (Matsui et al. 2004). DMKN beta splicing isoform was shown to inhibit activating phosphorylation of MAPK1/MAPK3, which depends on the interaction of DMKN with GRP78 (Higashi et al. 2012). The pups of mice deficient in Dmkn beta have smaller keratohyalin granules and their cornified envelopes are more sensitive to mechanical stress (Leclerc et al. 2014).

In human skin biopsies, FLG (filaggrin) is expressed in all keratinocytes above the stratum spinosum (Smoller et al. 1990), including keratinocytes of the granular layer of epidermis (Juhlin et al. 1992). In newborn mouse skin, FLG is first detected in large, irregular shaped keratohyalin granules in the keratinocytes of stratum granulosum, and then distributed throughout the cytoplasm of the corneocytes of the innermost layer of stratum corneum (Steven et al. 1990). Anti-(pro)filaggrin autoantibodies derived from patients with rheumatoid arthritis produce granular staining of stratum granulosum (Girbal Neuhauser et al. 1997). FLG is detectable in the cytosol and nucleus of stratum granulosum cells formed by cultured human keratinocytes. As FLG undergoes proteolytic cleavage upon terminal differentiation of granular keratinocytes, FLG cleavage products are detectable (Yoneda et al. 2012). Profilaggrin is expressed in the stratum granulosum of human epidermis by immunochemistry (Sakabe et al. 2013). Hypoxia leads to HIF1A- and EPAS1 (HIF2A)-mediated stimulation of FLG transcription in a Ca2+-independent and cell confluency-independent manner, and stimulates expression of SPINK5, an inhibitor of epidermal serine proteases that regulates conversion of profilaggrin into monomeric filaggrin. Three putative hypoxia response elements (HREs) are found in the promoter region of the human FLG gene, of which two HREs are functional. One of the two functional HREs is evolutionarily conserved. Direct binding of HIF1A/EPAS1 to these HREs has not been demonstrated. Simultaneous HIF1A and EPAS1 deficiency inhibits terminal differentiation of keratinocytes and formation of stratum granulosum and cornified envelope. ARNT Aryl hydrocarbon receptor nuclear translocator involved in ceramide synthesis also positively regulates FLG expression (Wong et al. 2015). Profilaggrin, a protein of >400 kDa that is the major protein constituent of keratohyalin granules in keratinocytes of stratum granulosum, consists of 10 to 12 near identical repeats of FLG monomers connected by short cleavable linkers. When granular keratinocytes commit to differentiation into corneocytes, profilaggrin is cleaved and 37 kDa FLG monomers are release. FLG monomers bind to and condense keratin cytoskeleton, facilitating cellular compression. In the stratum corneum, FLG monomers are further broken down to form natural moisturizing factor - NMF - a pool of hygroscopic amino acids and derivatives. Loss-of-function mutations in the FLG gene, present in ~10% of human population, are the cause of ichthyosis vulgaris, dry flaky skin phenotype. Ichthyosis vulgaris is mild in heterozygous carriers, and the phenotype severity also depends on environmental and lifestyle factors, being more pronounced in cold, dry climates, and ameliorated by the use of skin moisturizers. A naturally occurring Flg mutation in mice results in the "flaky tail" phenotype. Loss-of-function FLG mutations as well as subtle size variations in the FLG gene predispose carriers to atopic conditions such as eczema and allergy, by compromising the skin barrier and allowing allergens to cross the skin epidermis, triggering Th-2 allergic immunity (reviewed in McLean 2016). Profilaggrin, synthesized in granular keratinocytes, is proteolyzed into FLG monomers in cornified keratinocytes, concurrent with disappearance of nucleus and cell organelles and FLG mediated aggregation of keratin filaments into a tight matrix (reviewed in Ishida Yamamoto et al. 2018).

GJB3 (Gap junction beta 3 protein, also known as Connexin 31 or CX31), is expressed in stratum granulosum of mouse epidermis (Butterweck et al. 1994). In normal human epidermis, GJB3 is predominantly expressed in stratum granulosum, with a punctate staining at the plasma membrane, consistent with its involvement in formation of gap junctions. Mutations in GJB3 gene are associated with erythrokeratoderma variabilis (Di et al. 2001).

HOPX is an atypical homeodomain protein unable to bind to DNA, expressed in keratinocytes of stratum granulosum of human epidermis, and induced during Ca2+-mediated differentiation of cultured primary human keratinocytes. Overexpression of HOPX leads to upregulation of FLG and LORICRIN, while HOPX knockdown interferes with Ca2+-mediated induction of FLG and LORICRIN expression. Flg and Loricrin mRNA levels are downregulated in the epidermis of Hopx knockout mice (Obarzanek Fojt et al. 2011).

IVL is detected in stratum granulosum in normal epidermis and all layers except the basal layer in psoriatic epidermis (Bernard et al. 1985). In human skin biopsies, involucrin is expressed in all keratinocytes above the stratum spinosum (Smoller et al. 1990). Human IVL is expressed in keratinocytes of stratum granulosum, with expression increasing as the cells reach confluence (van Duijnhoven et al. 1992). In the keratinocytes of stratum granulosum, IVL is detected along the plasma membrane. In the corneocytes, involucrin labeling is reduced, but it is very intense in the psoriatic epidermis. Formation of the cornified envelope likely first involves the deposition of IVL, followed by incorporation of LORICRIN. In psoriatic epidermis, IVL deposition at the cornified envelope occurs precociously, without further maturation of the cornified envelope (Ishida Yamamoto et al. 1996). The distal regulatory region of the human IVL gene promoter, located at 2 2.5 kb upstream of the transcription start site, is needed to recapitulate the normal pattern of IVL expression in the epidermis of transgenic mice. The distal regulatory region contains two distinct, spatially separate regulatory elements that likely bind AP1 transcription factors (Crish et al. 2002). In the normal skin, IVL is detectable in the stratum granulosum and upper layers of stratum spinosum. IVL expression may be upregulated by IL13, IL17A, EDN1, TNF, IFNG, and MAPK1/MAPK3 and GSK3B signaling (Chen et al. 2013).

In normal epidermis, KLK5 (Kallikrein-5) and KLK7 (Kallikrein-7) are found in lamellar granules in stratum granulosum, localized separately from SPINK5, which is also found in lamellar granules (Ishida Yamamoto et al. 2005). KLK5 is expressed in stratum granulosum of human epidermis by immunochemistry. KLK5 co localizes with profilaggrin and is responsible for profilaggrin cleavage at the linker domain and release of FLG monomers (Sakabe et al. 2013). KLK5 and KLK7 co-localize with PRSS3 in the upper layers of stratum granulosum and can be activated by PRSS3 mediated proteolytic cleavage. KLK5 is thought to be the initiator of the kallikrein proteolytic cascade that leads to activation of kallikrein-related peptidases - KLKs - and degradation of corneodesmosomes, leading to desquamation - shedding of the outermost layer of corneocytes (Miyai et al. 2014).

Expression of KPRP gene, encoding human keratinocyte proline rich protein, is induced by Ca2+ in cultured normal human epidermal keratinocytes. KPRP is predominantly expressed in upper layers of stratum granulosum. In psoriatic epidermis, KPRP levels are increased compared to normal epidermis (Lee et al. 2005). KPRP is expressed in upper layers of stratum granulosum of human epidermis and is significantly decreased in atopic dermatitis compared to normal skin. KPRP co localizes with LORICRIN and is mainly detected in the cytoskeleton fraction of human keratinocytes (Suga et al. 2019). In an in vitro model of Ca2+ induced differentiation of human keratinocytes, KPRP is a marker of stratum granulosum, consistent with its in vivo expression pattern (Jeriha et al. 2020).

While keratinocytes in stratum granulosum stop transcribing keratin genes, keratin proteins, including KRT1, are still detectable (Totsuka et al. 2017, reviewed in Fuchs 1990).

KRT10 protein is detectable in stratum granulosum keratinocytes (Totsuka et al. 2017). In stratum granulosum formed by cultured human keratinocytes, KRT10 protein is detectable in the cytosol and nucleus, and co-localizes with the N-terminal cleavage product of profilaggrin (Yoneda et al. 2012).

KRTDAP gene, encoding keratinocyte differentiation-associated protein (also known as KDAP), is part of the keratinocyte related locus on human chromosome 19q13.1 that also includes SBSN and DKMN genes. Coordinated transcription of these genes is detectable in stratum spinosum, but proteins are the most prominent in the granular layer keratinocytes, from which they are secreted (Matsui et al. 2004). In normal human skin, KRTDAP protein is found exclusively in lamellar granules of keratinocytes in stratum granulosum and in the intercellular space in stratum corneum. In psoriatic skin, KRTDAP is expressed more widely and detected in all suprabasal keratinocytes (Tsuchida et al. 2004).

In newborn mouse skin, LORICRIN is first detected in small, round, keratohyalin granules in keratinocytes of stratum granulosum, and then at the periphery of corneocytes throughout the stratum corneum (Steven et al. 1990). LORICRIN is expressed in keratinocytes of granular layer of human epidermis. In skin disorders characterized by incomplete differentiation, e.g. psoriasis, LORICRIN is not detected but FLG is still expressed (Juhlin et al. 1992). LORICRIN mRNA and protein are present in stratum granulosum and both transcript and protein are diminished upon treatment of human epidermis culture with retinoic acid (Magnaldo et al. 1992). LORICRIN is distributed diffusely in keratinocytes of stratum granulosum, occasionally in granular aggregates in the cytoplasm and nucleus, and concentrated on the desmosomal attachment plaques where it co-localizes with desmoglein. LORICRIN is diminished in psoriatic epidermis (Ishida Yamamoto et al. 1996). LORICRIN, diffusely distributed in granular keratinocytes, is associated with the cornified envelope in corneocytes, a tough structure beneath the corneocyte plasma membrane. Mutations in LORICRIN gene are implicated in two hereditary skin disorders, Vohwinkel's syndrome and progressive symmetric erythrokeratoderma (reviewed in Ishida Yamamoto et al. 1998). LORICRIN is detectable in the cytosol and nucleus of cultured human keratinocytes in stratum granulosum, where it co-localizes with the N-terminal cleavage product of FLG (Yoneda et al. 2012). As granular keratinocytes differentiate into corneocytes, cornified envelope is formed by covalent crosslinking through transglutamination of various proteins, including LORICRIN, which is one of the main constituents of the cornified envelope (reviewed in Ishida Yamamoto et al. 2018).

PRSS3, also known as mesotrypsin, is expressed only in the granular layer of epidermis. In cultured keratinocytes, PRSS3 mRNA is expressed at the confluent stage and is strongly upregulated after air exposure (Nakanishi et al. 2010). PRSS3 is expressed in the upper granular layer of epidermis, where KLKs and SPINK5 are also found. PRSS3 is able to proteolytically activate KLK5 and KLK7, and hydrolyze SPINK5 fragments bound to KLKs, thus contributing to the desquamation process. PRSS3 expression level in epidermis decreases after the age of 60 (Miyai et al. 2014).

SBSN gene, encoding Suprabasin, is part of the keratinocyte related locus on human chromosome 19q13.1 that also includes KRTDAP and DKMN genes. Coordinated transcription of these genes is detectable in stratum spinosum, but proteins are the most prominent in the granular layer keratinocytes, from which they are secreted (Matsui et al. 2004). The cluster of Sbsn, Krtdap, and Dkmn genes is conserved on mouse chromosome 7, and these genes are coordinately expressed during epidermal differentiation (Bazzi et al. 2007). SBSN deficient human skin three dimensional model has compact stratum corneum, immature stratum granulosum, and increased keratinocyte apoptosis (Aoshima et al. 2019).

SPINK5, also known as serine protease inhibitor LEKTI, is strongly expressed in keratinocytes of stratum granulosum and uppermost layers of keratinocytes of stratum spinosum. Two isoforms of SPINK5, of 145 kDa and 125 kDa, are detectable in keratinocytes. Both isoforms are glycosylated ad processed by a FURIN-like enzyme in the post-endoplasmic reticulum compartment, yielding fragments of 42 kDa, 65 kDa, and 68 kDa. SPINK5 genetic defects lead to Netherthon syndrome, a severe autosomal recessive ichtyosis (Bitoun et al. 2003). In normal epidermis, SPINK5 is found in lamellar granules, membrane-bound organelles of stratum granulosum and uppermost layers of stratum spinosum, that fuse with the plasma membrane and secrete their contents into the extracellular space. SPINK5 localizes separately from KLK5 and KLK7, which are also found in lamellar granules, and is likely secreted before KLK5 and KLK7 (Ishida Yamamoto et al. 2005). SPINK5 is expressed in keratinocytes of stratum granulosum of both human and mouse epidermis (Galliano et al. 2005). SPINK5 cleavage fragments function as inhibitors of KLK5 and KLK7 proteases, preventing premature degradation of corneodesmosomes by KLK5/KLK7, and thus maintaining integrity and cohesion of the stratum corneum (reviewed in Ishida Yamamoto et al. 2018).

SPRR2A gene is part of a gene cluster of 1.5 Mb on human chromosomal band 1q21 that includes several other small proline-rich protein genes, as well as LORICRIN and IVL genes, which have all likely evolved from a common ancestor. By immunohistochemistry, SPRR2A protein is expressed at moderate levels in stratum granulosum of interfollicular epidermis, and by in situ RNA hybridization SPRR2A mRNA is restricted to the uppermost spinous and granular epidermis layers. Retinoic acid inhibit accumulation of SPRR2A mRNA and protein in keratinocytes. SPRR2A may be a precursor protein of the cornified envelope (Hohl et al. 1995). SPRR2A expression is strongly induced by binding of the epithelium-specific transcription factor ELF3, also known as ESE-1, upregulated during Ca2+-induced terminal differentiation of keratinocytes, to the Ets site in the SPRR2A gene promoter (Sark et al. 1998). POU2F3 transcription factor isoform Skn-1a cooperates with the transcription factor ELF3 in the induction of SPRR2A gene expression during terminal differentiation of keratinocytes in the epidermis (Cabral et al. 2003).

Markers of the stratum corneum keratinocytes (corneocytes) are summarized in the "Table of markers of corneocytes in interfollicular epidermis". Some of the markers, in particular LGLAS7, are described in more detail in the section "Keratinocyte stem cell differentiates into transit amplifying cell in the basal layer of interfollicular epidermis".


Table of markers of corneocytes in interfollicular epidermis. Please note that keratinocytes in CellMarker database and PanglaoDB correspond to corneocytes in Reactome.

Marker (protein/RNA)	Literature Reference	CellMarker database – RNA/Protein (Hu et al. 2022)	PanglaoDB – RNA (Franzén et al. 2019)
CALML5 (protein)	Mehul et al. 2001 Mehul et al. 2006	No	Yes
CASP14	Fischer et al. 2004 Alibardi et al. 2004	No	No
FLG	Simon et al. 1996 Girbal Neuhauser et al. 1997 Pendaries et al. 2014	No	Yes
KLK5	Caubet et al. 2004 de Veer et al. 2017	No	No
KLK7	Caubet et al. 2004 de Veer et al. 2017	No	No
LGALS7	Magnaldo et al. 1995 Umayahara et al. 2020	No	Yes

CALML5 is expressed in keratinocytes of lower layers of the stratum corneum. CALML5 is proteolytically degraded in upper layers of normal but not psoriatic stratum corneum, which is prevented by Ca2+ (Mehul et al. 2006).

CASP14 is highly expressed in differentiated epidermal keratinocytes and is the predominant caspase in the stratum corneum where it is normally present exclusively in its processed form. Both pro-caspase-14 and CASP14 subunits produced by cleavage are present in incompletely matured stratum corneum in psoriasis and seborrheic dermatitis (Fischer et al. 2004). CASP14 is first detectable in the nuclei and keratohyalin granules of stratum granulosum keratinocytes, and it is present in the cornified envelope, cytosol, and nucleus of corneocytes (Alibardi et al. 2004).

FLG partially co-localizes with the cornified envelope in corneocytes (Simon et al. 1996). Anti-(pro)filaggrin autoantibodies derived from patients with rheumatoid arthritis produce diffuse staining of stratum corneum (Girbal-Neuhauser et al. 1997). FLG is expressed in corneocytes and needed for the maintenance of the corneocyte intracellular matrix.

KLK5 (Caubet et al. 2004). KLK5 is expressed in stratum corneum and is able to directly cleave all three components of corneodesmosomes, CDSN, DSC1, and DSG1, and to proteolytically activate KLK7; de Veer et al. 2017: KLK5 can be isolated from the plantar stratum corneum, and contributes to desquamation).

KLK7 is expressed in the stratum corneum and can directly cleave CDSN and DSC1 components of corneodesmosomes, but not DSG1 (Caubet et al. 2004). KLK7 can be isolated from the plantar stratum corneum and is the main protease involved in desquamation (de Veer et al. 2017).

LGALS7 is expressed in all epidermal layers, especially highly in the stratum corneum (Umayahara et al. 2020).

For molecular details of cornified envelope formation during differentiation of stratum granulosum keratinocytes into corneocytes, please refer to Reactome pathway "Formation of the cornified envelope", a subpathway of the Reactome "Keratinization" pathway (Blumenberg, M. 2016. “Keratinization.” Reactome - a Curated Knowledgebase of Biological Pathways 58 (September). https://doi.org/10.3180/r hsa 6805567.1).