Transient Expression of Epidermal Filaggrin in Cultured Cells Causes Collapse of Intermediate Filament Networks with Alteration of Cell Shape and Nuclear Integrity.

Abstract

Filaggrin is an intermediate filament-associated protein (IFAP) that aggregates epidermal keratin filaments in vitro and is thought to perform a similar function during terminal differentiation in vivo. To test this function in living cells, we transiently expressed constructs encoding human filaggrin in both simple epithelial cells (COS-7) and rat keratinocytes. Scanning laser confocal microscopy showed that filaggrin-positive cells had collapsed keratin and vimentin intermediate filament (IF) networks, and that filaggrin partially co-localized with the IF networks. Filaggrin was also detected diffusely in the cytoplasm and nucleus. In contrast, when profilaggrin-like constructs, containing five filaggrin domains separated by the linker sequences, were expressed in cultured cells, immunoreactive granules formed. This finding is reminiscent of the insoluble nature of native profilaggrin that accumulates in keratohyalin granules in vivo, suggesting that the linker peptides (present in profilaggrin but not filaggrin) are important for granule formation. Cells expressing filaggrin also displayed disruption of the nucleus and the nuclear envelope; they rounded up and lost attachment to the substratum, in contrast to control cells over-expressing beta-galactosidase. This functional test of filaggrin in living cells supports its role in the reorganization and packing of keratin IF in epidermal differentiation. Moreover, the observed effects on cell morphology and nuclear integrity suggest that filaggrin may contribute to the form of apoptosis associated with terminal differentiation in epidermis.

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

“…These observations are consistent with the known role of FLG in keratin intermediate filament aggregation. 40,80 Although the role for FLG in intermediate filament aggregation in IV has been disputed, 81 FLG mutation testing was unavailable at that time. Moreover, keratin intermediate filament integrity, assessed by susceptibility to urea extraction, remains unchanged in the organotypic, siRNA knock-down cultures that we used in these studies.…”

“…Alternatively, dominant-negative disruption of the cytoskeleton, as occurs with mutations in either keratin 1 or 10 in epidermolytic ichthyosis (epidermolytic hyperkeratosis), blocks lamellar body (LB) secretion, also resulting in a downstream, paracellular permeability barrier defect. 36 Since FLG peptides normally associate with both the CE [37][38][39] and keratin filaments, 40 FLG deficiency could compromise permeability barrier function by either mechanism, by both mechanisms, or by a mechanism that is unique to IV. Although in the pre-genotype era, barrier function was shown to be moderately abnormal in IV and AD patients, [41][42][43][44][45] this has not been assessed in genotyped, FLG-deficient IV patients, and there is little evidence for the mechanisms of a potential barrier defect.…”

AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation

“…Filaggrin has been shown to be associated with nuclear degradation and keratin intermediate filament aggregation. 28,29 Studies have reported that keratohyalin granules with profilaggrin aggregates localise to the perinuclear region sometimes forming deep invaginations at the areas of association. 28,29 This is thought to facilitate entry of cleaved N-terminal domain of profilaggrin into the nucleus, where it favours nuclear degradation.…”

“…28,29 Studies have reported that keratohyalin granules with profilaggrin aggregates localise to the perinuclear region sometimes forming deep invaginations at the areas of association. 28,29 This is thought to facilitate entry of cleaved N-terminal domain of profilaggrin into the nucleus, where it favours nuclear degradation. 28 One potential explanation for the altered processing of filaggrin, partially supported by our data, is that filaggrin interacts with nuclear lamins and therefore the persistence of Lamin A/C increases its association with filaggrin intermediates at the nuclear lamina, preventing its processing to more mature forms.…”

“…28,29 This is thought to facilitate entry of cleaved N-terminal domain of profilaggrin into the nucleus, where it favours nuclear degradation. 28 One potential explanation for the altered processing of filaggrin, partially supported by our data, is that filaggrin interacts with nuclear lamins and therefore the persistence of Lamin A/C increases its association with filaggrin intermediates at the nuclear lamina, preventing its processing to more mature forms. Alternatively, the observation of nuclear filaggrin in the S404A-and S301A-expressing cells could be due to the non-degraded nuclei preventing proper access to the protein by cytoplasmic proteases.…”