Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR (original) (raw)
Related papers
Basement membranes in skin: unique matrix structures with diverse functions?
Histochemistry and Cell Biology, 2009
The view of extracellular matrix (ECM) has evolved from a merely scaVolding and space Wlling tissue element to an interface actively controlling cellular activities and tissue functions. A highly specialized form of ECM is the basement membrane (BM), an ubiquitous sheet-like polymeric structure composed of a set of distinct glycoproteins and proteoglycans. In this review we are largely focusing on function and assembly of BM in skin (1) at the dermo-epidermal interface and (2) in the resident microvasculature. The role of the non-polymeric components perlecan and particularly nidogen is exempliWed by reviewing experiments based on genetic approaches and adequate experimental skin models in vivo and in vitro. While in mice total deWciency of one of these components is eventually developmentally lethal, the severity of the defects varies drastically between tissues and also the skin models recapitulating BM formation in vitro. There is accumulating evidence that this relies on the mechanical properties, the molecular composition of the BM, the adjacent ECM or connective tissue, the dynamics of molecular assembly, and 'minor' tissue-speciWc modiWer or adapter components. Though the role of nidogen or perlecan is still remaining a controversial issue, the statements 'being essential for BM/or not' should be consequently referred to the developmental, tissue, and functional (e.g., repair) context.
Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells
The Journal of Cell Biology, 1982
Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors.
PROTEOMICS, 2006
The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.
Differentiation, 1997
Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains α2, α3, β1 and α6, while β4 already showed a preferential basal location; (2) de novo expression of α5 and αv; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular α2β1 and α3β1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin α6β4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidation. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.
Epidermal Basement Membrane Substitutes for Bioengineering of Human Epidermal Equivalents
JID Innovations, 2021
Epidermal basement membrane, a tightly packed network of extracellular matrix (ECM) components, is a source of physical, chemical, and biological factors required for the structural and functional homeostasis of the epidermis. Variations within the ECM create distinct environments, which can affect the property of cells in the basal layer of the epidermis and subsequently affect keratinocyte differentiation and stratification. Very little attention has been paid to mimicking basement membrane in organotypic cultures. In this study, using parameters outlined in a consensus on the quality standard of organotypic models suitable for dermatological research, we have evaluated three basement membrane substitutes. We compared fibronectin with three complex three-dimensional matrices: Matrigel, decellularized dermal fibroblast-produced and-assembled ECM, and a dry human amniotic membrane. Our results suggest that Matrigel is not a suitable substrate for human epidermal equivalent culture, whereas the two other complex three-dimensional substitutes, decellularized dermal fibroblast-produced and-assembled ECM and dry human amniotic membrane, were superior to single layer fibronectin coating. Human epidermal equivalents cultured on either decellularized dermal fibroblast-produced and-assembled ECM or on dry human amniotic membrane generated hemidesmosomes, whereas those on fibronectin did not. In addition, human epidermal equivalent cultured on decellularized dermal fibroblast-produced and-assembled ECM and on dry human amniotic membrane can be maintained in culture 4 days longer than human epidermal equivalent cultured on fibronectin without compromising the barrier function.
Journal of Dermatological Science, 1998
To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and α3, β1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of α2, α3, α6, β1, and β4 integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the β1 (AIIB2) and α6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin γ1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin γ2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a selfassembly process.
[Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins]
Tsitologiia, 2006
Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading c...
Burns, 1996
Previous studies have demonstrated that components of the extracellular matrix can induce neurite extension and cell adhesion in the neuroblastoma × glioma hybrid cell line, NG108-15. Using standard intracellular recording techniques, we examined the resting membrane potential (RMP) and membrane excitability of NG108-15 cells differentiated under serum-free media with representative extracellular matrix (ECM) protein components as the substrate. Surfaces coated with collagen IV and a laminin-1 synthetic peptide induced a significantly (P Ͻ 0.05) more hyperpolarized RMP than control polystyrene surfaces. For example, after ≥8 days in culture NG108-15 cells plated on polystyrene exhibited a RMP of −33.2 ± 0.8 mV (mean ± SEM, n = 158 cells) whereas cells cultured on the laminin-1 peptide C16 and collagen IV showed a RMP of −37.6 ± 0.7 mV (n = 157) and −37.5 ± 1.5 mV (n = 68), respectively. Furthermore, the proportions of cells on ECM substrates showing membrane excitability, i.e. evoked action potentials (APs) and the capability for regular firing, were significantly greater compared to those cells cultured on polystyrene. Among excitable cells cultured on the different substrates, characteristics of the action potentials, such as AP duration, amplitude, and the maximum rate of rise, dV/dt MAX , were examined in detail. While little or no differences were observed between polystyrene and the laminin-1 peptide groups, significant differences in the AP parameters were apparent for collagen IV. For example, dV/dt MAX for polystyrene and the laminin-1 peptide C16 were only 71.7 ± 24.5 V/s (n = 11) and 59.0 ± 8.9 V/s (n = 9), respectively, whereas cells cultured on collagen IV surfaces exhibited a dV/dt MAX reaching 156.1 ± 22.0 V/s (n = 7). These data support a role for ECM components in the maintenance of the RMP and membrane excitability in NG108-15 cells.