Mechanotransduction of keratinocytes in culture and in the epidermis

The epidermis, like many other tissues, reacts to mechanical stress by increasing cell proliferation. Mechanically stressed skin regions often develop thicker skin and hyperkeratosis. Interestingly, a large number of skin diseases are accompanied by epidermal proliferation and hyperkeratosis even under normal mechanical stress conditions. Although, some of the molecular pathways of mechanical signaling involving integrins, the epidermal growth factor receptor and mitogen-activated protein kinases are known it is still unclear, how mechanical force is sensed and transformed into the molecular signals that induce cell proliferation. This review focuses on the molecules and pathways known to play a role in mechanotransduction in epidermal keratinocytes and discusses the pathways identified in other well-studied cell types.     

Epidermal Growth Factor and EGF Receptors are mainly expressed in the wound epidermis and proliferating ependyma of the regenerating tail of lizards

The presence of EGF and its receptor during tail regeneration in lizard has been assessed by immunoblotting and immunofluorescence to test whether this growth factor may be involved in the process. Immunolabelled bands at 8 and 42–46 kDa for EGF are detected in the regenerating tail. A main band at 45–50 kDa and other weaker bands at lower or higher molecular weight for the EGF receptor are also present. The results indicate that degraded forms of the protein are present although the specific nature of the different bands could not be determined. Immunofluorescence indicates that EGF-labelled cells and EGF receptor are especially seen in the wound epidermis and in the cytoplasm of ependymal cells. Numerous basal keratinocytes of the wound epidermis and apical epidermal peg contain labelled nuclei for EGFR, suggesting that activated receptor stimulates intense cell proliferation of the wound epidermis. Blastema and labelled myoblasts are occasionally detected in early differentiating muscles, but almost no labelled chondroblasts are present in the differentiating cartilaginous tube. The study indicates that EGF and its receptor are mainly present in epithelial cells in a form that allows them to regulate proliferation during tail regeneration.     

Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/β-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active β-catenin, two key members of the Wnt/β-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/β-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.     

Inhibition of autonomous human keratinocyte proliferation and amphiregulin mitogenic activity by sulfated polysaccharides

Summary We previously demonstrated that human keratinocyte cultures proliferate in the absence of polypeptide growth factors (autonomous growth) and that this autonomous growth is blocked by interaction of heparin with a human keratinocyte-derived autocrine factor (KAF) which we identified as amphiregulin (AR). In the present study, we demonstrate that sulfated polysaccharides other than heparin (low and high molecular weight dextran sulfates) also inhibit the AR-mediated autonomous proliferation of human keratinocytes. Furthermore, sulfated polysaccharides such as high and low molecular weight dextran sulfates, heparan sulfate and, to a lesser extent, chondroitin sulfates B and C were also shown to be inhibitors of human keratinocyte-derived AR (k-d AR)-stimulated DNA synthesis in quiescent murine AKR-2B cell cultures. Our results demonstrate that sulfation of polysaccharides is required for AR inhibitory activity, and that several sulfated polysaccharides (other than heparin) can act as inhibitors of AR-mediated autonomous proliferation in human epidermal keratinocytes and as inhibitors of k-d AR-mediated mitogenic activity in AKR-2B cells.     

Zinc accumulation and metallothionein gene expression in the proliferating epidermis during wound healing in mouse skin

Metallothionein (MT), a low molecular weight metal-binding protein, has been related to zinc and copper metabolism, the acute-phase response, and cellular proliferation. In this study, we investigated changes in zinc metabolism and MT gene expression occurring in tissue damage and repair during wound healing in mouse skin. Northern blot analysis revealed that a significant increase of MT mRNA was observed in the liver for 18 h after wounding, and serum zinc downfall and hepatic zinc uptake were observed. In situ hybridization analysis showed that no significant expression of MT mRNA was detected within the first 9 h after wounding. However, it was expressed restrictively in the proliferating epidermis of the wound margin after 12 h. Zinc began to accumulate in wounded skin after MT gene expressed. Northern blotting and immunocytochemical staining revealed that MT has been synthesized actively during the growth phase compared with the stationary phase in normal human epidermal keratinocytes in vitro. Intracellular zinc accumulation was observed in the proliferating cells. We concluded that hepatic MT plays an important role as an acute phase protein against host damage, and epidermal MT contributes in the supply of zinc to wounded tissue and activates proliferation for the regeneration of epidermis. Accepted: 2 July 1999     

Effects of a Synthetic bFGF Antagonist Peptide on the Proteome of 3T3 Cells Stimulated with bFGF

It has been known that basic fibroblast growth factor (bFGF) plays an important role in tumor progression mainly due to its strong mitogenic activity. Antagonists targeting bFGF have been considered a potential strategy for cancer therapy via inhibiting cell proliferation induced by bFGF. We have previously obtained a high-affinity bFGF-binding peptide (named as P7) with strong inhibitory activity against bFGF-induced cell proliferation from the phage display random heptapeptide library. The aim of the present investigation was to synthesize the peptide P7 by solid phase method and explore the mechanisms of the inhibitory effect of P7-targeting bFGF stimulation on Balb/c 3T3 cells via proteomic analysis. Seven differentially expressed protein were identified, among which four were decreased by bFGF stimulation alone and increased by addition of P7, the other three were up-regulated by bFGF treatment alone and down-regulated by addition of P7. Among the identified proteins, epidermal fatty acid-binding protein (E-FABP) and low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) take part in the regulation of cell proliferation. The results suggested P7 inhibited the bioactivities of bFGF possibly via influencing the expression of cellular proteins related to cell proliferation.     

Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner

Adiponectin, an adipocyte-specific secretory protein, is present in serum as three oligomeric complexes. Apart from its roles as an anti-diabetic and anti-atherogenic hormone, adiponectin has been implicated as an important regulator of cell growth and tissue remodeling. Here we show that some of these functions might be mediated by the specific interactions of adiponectin with several important growth factors. Among six different growth factors examined, adiponectin was found to bind with platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (FGF), and heparin-binding epidermal growth factor-like growth factor (HB EGF) with distinct affinities. The bindings of adiponectin with these growth factors are oligomerization-dependent. PDGF-BB bound to the high molecular weight (HMW) and middle molecular weight (MMW) complexes, but not to the low molecular weight (LMW) complex of adiponectin. Basic FGF preferentially interacted with the HMW form, whereas HB EGF bound to all three forms with comparable affinities. These three growth factors did not compete with each other for their bindings to adiponectin, suggesting the involvement of distinct binding sites. The interactions of adiponectin with PDGF-BB, basic FGF, and HB EGF precluded the bindings to their respective membrane receptors and attenuated the DNA synthesis and cell proliferation induced by these growth factors. Small interfering RNA-mediated down-regulation of adiponectin receptors did not affect the suppressive effects of adiponectin on cell proliferation stimulated by these growth factors. These data collectively suggest that the oligomeric complexes of adiponectin can modulate the biological actions of several growth factors by controlling their bioavailability at a pre-receptor level and that this effect might partly account for the anti-atherogenic, anti-angiogenic, and anti-proliferative functions of adiponectin.     

Body-specific proliferation of adult precursor cells in Xenopus larval epidermis

Cell proliferation was examined in the back and tail epidermis of larval Xenopus laevis using bromodeoxyuridine (BrdU). The BrdU labeling index of the back epidermis increased temporally at stage 59, followed by a rapid decrease to the same level as at stage 51. The temporal increase in cell proliferation of the back epidermis produced a new epidermal layer composed of basal cells. In vitro analysis showed that tri-iodothyronine (T₃) promotes cell proliferation of basal cells but suppresses that of skein cells. Immunohistochemical studies showed that the newly formed basal cell layer functions as adult precursor cells which produce the adult epidermal cells. In contrast to the back epidermis, the labeling index of the tail epidermis decreased from stage 57. However, when the tail skin was transplanted to the back area, cell proliferation in the tail epidermis increased to the same level as that of the normal back epidermis. Cell proliferation of the back epidermis was not suppressed by transplanting the skin to the tail area. These results suggest that some promoting factors are produced in the body region and regulate the number of adult precursor cells, which determine the developmental fate of the larval skin.     

Biochemical and immunological analysis of a basic protein from newborn rat epidermis.

A basic protein, solubilized in buffered salt solutions from keratohyalin granules of newborn rat epidermis, has been purified by ion-exchange chromatography. The relative molecular weight of the protein was determined as 12 800 +/- 200 from its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is relatively rich in lysine, glycine, alanine, and valine which together comprise about 60% of the total amino acid residues. Using an antibody to this protein, which we have designated fraction 4, we have found that it is specific to rat epidermis and is not present in any other rat tissues or in epidermal extracts from other species. The cells of the four epidermal layers were separated and the amount of fraction 4 in each cell layer was measured by radioimmunoassay. The protein is localized mainly in the upper layers of epidermis. The protein, which binds to DNA, appears in the epidermis just prior to birth, increases during the first week of post-natal life and declines sharply thereafter. Fraction 4 represents about 7% of the total solubilized protein in 7-day-old rat epidermis.     

Effects of low molecular weight soybean peptide on mRNA and protein expression levels of differentiation markers in normal human epidermal keratinocytes

Low molecular weight soybean peptide (LSP) was applied to normal human epidermal keratinocytes, and the results showed a significant increase in the gene expression levels of involucrin, transglutaminase, and profilaggrin. Filaggrin protein levels were also significantly higher. It is possible that LSP has an epidermal cell differentiation-promoting effect and may be able to regulate metabolism of the epidermis.     

Pathways sufficient to induce epidermal carcinogenesis

Abnormal epidermal proliferation is characteristic of a number of disorders, including the two most common cancers in the United States, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Both cancers display a disruption in the normal homeostatic balance between cell division and programmed cell death. While abnormal activation of the sonic hedgehog/patched pathway has been established as sufficient to induce hallmark features of BCC in both human and murine epidermis, pathways sufficient to convert normal epidermis into SCC have been less well defined. Building on findings that indicate a potent role for Ras and NF-kappaB in normal epidermal growth regulation, recent work indicates that activation of Ras signaling in concert with inhibition of NF-kappaB function is entirely sufficient to transform normal human epidermis into tumor tissue with all the cardinal features of SCC.     

Hormonal regulation of c-erbB-2 oncogene expression in breast cancer cells.

Expression of the c-erbB-2 (neu, HER-2) oncogene is found to be subjected to hormonal and developmental regulation in normal as well as neoplastic mammary cells. We have previously reported that estrogens inhibit c-erbB-2 expression at both the mRNA and protein level in estrogen receptor (ER)-positive, but not in ER-negative, breast cancer cell lines. Reversion of c-erbB-2 inhibition is seen with tamoxifen. The effect on c-erbB-2 expression of several other hormones and factors, which influence mammary cell growth and differentiation, has been studied. Our observations indicate that, in normal and neoplastic mammary cells, c-erbB-2 expression is inversely related to cell proliferation. While estrogens, anti-estrogens and cAMP clearly regulate c-erbB-2 mRNA levels, epidermal growth factor dramatically decreases the c-erbB-2 protein without affecting the level of c-erbB-2 mRNA. Therefore, different signals converging in terms of cell proliferation regulate c-erbB-2 expression by different molecular mechanisms.     

The proteins of the embryonic chick epidermis : I. During the normal development in ovo

As a preliminary to a study of the proteins of the embryonic chick epidermis when grown in vitro under various culture conditions, the proteins of the anterior metatarsal epidermis, from 11 days of embryonic life up to 9 days posthatching, have been studied. Carboxymethylated derivatives of the proteins extracted by a thiol reduction procedure have been analyzed by polyacrylamide gel electrophoresis. The results have shown that the differentiation of the epidermis is characterized by the appearance between days 14 and 17 of at least 11 major protein bands in the electrophoretic pattern. Two of these bands are of relatively high molecular weight protein and appear earlier than the remaining bands which form a group of closely related, low molecular weight protein species. The differentiation of the tissue also involves the disappearance from the electrophoretic pattern of all but one of the five major bands present in extracts of the 11/12-day epidermis. A study of the proteins derived from the isolated periderm of the 14-day chick embryo beak has suggested that one of the major bands in the 11/12-day metatarsal epidermal extracts may be a peridermal protein.     

Pathways Sufficient to Induce Epidermal Carcinogenesis

Abnormal epidermal proliferation is characteristic of a number of disorders, including the two most common cancers in the United States, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Both cancers display a disruption in the normal homeostatic balance between cell division and programmed cell death. While abnormal activation of the sonic hedgehog/patched pathway has been established as sufficient to induce hallmark features of BCC in both human and murine epidermis,1-4 pathways sufficient to convert normal epidermis into SCC have been less well defined. Building on findings that indicate a potent role for Ras and NF-?B in normal epidermal growth regulation,5-9 recent work indicates that activation of Ras signaling in concert with inhibition of NF-?B function is entirely sufficient to transform normal human epidermis into tumor tissue with all the cardinal features of SCC.