The Expression of Psoriasin (S100A7) and CD24 Is Linked and Related to the Differentiation of Mammary Epithelial Cells

Psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is highly expressed in high-grade ductal carcinoma in situ (DCIS) and in the benign hyperproliferative skin disorder psoriasis. The gene that encodes psoriasin and many other S100 genes are located within a gene cluster on chromosome region 1q21, known as the epidermal differentiation complex. This cluster contains genes for several differentiation markers that play important roles in the terminal differentiation of the epidermis. The purpose of the present study was to evaluate the role of psoriasin in the differentiation process of mammary epithelial cells. Normal mammary epithelial cells (MCF10A) cultured in confluence and suspension, conditions known to induce psoriasin expression, demonstrated a shift towards a more differentiated phenotype indicated by an increase in the expression of the luminal differentiation markers CD24 and MUC1 and the reduced expression of the breast stem cell marker CD44. The expression of psoriasin and MUC1 was most pronounced in the CD24$^+$-enriched fraction of confluent MCF10A cells. The shift towards a more differentiated phenotype was abolished upon the downregulation of psoriasin using short hairpin RNA (shRNA) and small interfering RNA (siRNA). Using specific inhibitors, we showed that psoriasin and CD24 expression was regulated by reactive oxygen species (ROS) and the nuclear factor (NF)-κB signaling pathways. While immunohistochemical analyses of DCIS showed heterogeneity, the expression of psoriasin and CD24 showed a similar staining pattern. Our findings suggest that the expression of psoriasin is linked to the luminal differentiation marker CD24 in mammary epithelial cells. Psoriasin demonstrated an essential role in the shift towards a more differentiated CD24$^+$ phenotype, supporting the hypothesis that psoriasin plays a role in the differentiation of luminal mammary epithelial cells

Psoriasin For Better or for Worse in Sickness and in Health : The Role of Psoriasin in Angiogenesis and Differentation of Epithelial Cells

Psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is highly expressed in high-grade ductal carcinoma in situ (DCIS) and in the benign hyper-proliferative skin disorder psoriasis. Both breast cancer and psoriasis are diseases which are characterized by hyperproliferation and a disturbed differentiation of the epithelial cells as well as a pronounced angiogenesis. The potential role of psoriasin in angiogenesis and the epithelial differentiation remain unclear. The aim of this thesis was to investigate the cellular effects of psoriasin in angiogenesis and the differentiation processes, with special emphasis on breast cancer and psoriasis. We found that psoriasin expression was induced in mammary epithelial cells and keratinocytes by oxidative stress. Psoriasin expression was shown to induce vascular endothelial growth factor (VEGF) expression and several other pro-angiogenic factors in epithelial cells. Upon down-regulation of psoriasin, H2O2-induced expression of VEGF was decreased as well as the pro-angiogenic factors heparin-binding EGF-like growth factor (HBEGF) and matrix metalloproteinase (MMP)-1. Extracellular psoriasin contributed to the subsequent induction of proliferation, migration and tube formation of endothelial cells. The proliferative effect of psoriasin was shown to be mediated by the receptor for advanced glycation end products (RAGE). Furthermore, psoriasin induced reactive oxygen species (ROS) in both endothelial and epithelial cells through the action of RAGE, and contributed to the expression of the pro-angiogenic factors in endothelial cells. The expression of psoriasin was up-regulated in mammary epithelial cells and keratinocytes in response to differentiation-inducing stimuli and was shown to be regulated by pathways involved in epithelial cell differentiation. Upon psoriasin down-regulation the shift towards a more differentiated CD24+-phenotype of mammary epithelial cells was abolished. Furthermore, the expression of the differentiation markers involucrin, desmoglein 1, transglutaminase 1 and CD24 was decreased in keratinocytes upon down-regulation of psoriasin expression. In vivo we demonstrated a gradient of psoriasin expression in the psoriatic epidermis, with intense expression in the suprabasal differentiated layers, and a similar staining pattern between psoriasin and the differentiation marker CD24 in DCIS tumors. In conclusion, our findings describe psoriasin as a mediator in the angiogenic process and a contributor of epithelial cell differentiation. Consequently, psoriasin is possibly a contributor to the development and progression of breast cancer and psoriasis and a potential target in the treatment of these diseases

Overexpression of Psoriasin (S100A7) Contributes to Dysregulated Differentiation in Psoriasis.

Psoriasin, which is highly expressed in psoriasis, is encoded by a gene located within the epidermal differentiation complex. The aim of this study was to investigate the effect of endogenous psoriasin on disturbed keratinocyte differentiation in psoriasis. Immunohistochemical staining revealed a gradient of psoriasin expression in the psoriatic epidermis with highest expression in the suprabasal, differentiated layers. Induction of keratinocyte differentiation caused concurrent expression of psoriasin and the differentiation marker involucrin. The differentiation-induced psoriasin expression was found to be mediated by the protein kinase C pathway. The downregulation of psoriasin expression by small interfering RNA revealed that psoriasin mediates the expression of involucrin, desmoglein 1, transglutaminase 1 and CD24 in normal differentiation. The lentivirus-mediated overexpression of psoriasin, mimicking the psoriatic milieu, gave rise to an altered regulation of differentiation genes and an expression pattern reminiscent of that in psoriatic epidermis. These findings suggest that psoriasin contributes to the dysregulated differentiation process in the psoriasis epidermis.Funding agencies: Ingrid Asp Foundation; Welander Foundation; Swedish psoriasis association; Medical Research Council</p

Psoriasin (S100A7) increases the expression of ROS and VEGF and acts through RAGE to promote endothelial cell proliferation

Psoriasin (S100A7), originally identified in psoriasis, is a calcium-binding protein belonging to the multigenic S100 family. In high-grade ductal carcinoma in situ, psoriasin was identified as one of the most abundant transcripts. We have previously shown that psoriasin was induced by reactive oxygen species (ROS). Moreover, the downregulation of psoriasin by short hairpin RNA (shRNA) led to the reduced expression of vascular endothelial growth factor (VEGF) and inhibited tumor growth in vivo. The aim of the present study was to investigate whether psoriasin could have direct effects on endothelial cells. In this study we demonstrated that psoriasin increased VEGF expression in mammary epithelial cells. The treatment of endothelial cells with recombinant psoriasin increased proliferation comparable to that of recombinant VEGF protein. No change in proliferation was seen when endothelial cells were infected with psoriasin-expressing adenoviruses, suggesting that the proliferative effect of psoriasin was mediated by a specific receptor. Treatment with sRAGE, targeting the receptor for advanced glycation end products (RAGE), thus inhibited endothelial cell proliferation and tube formation enhanced by recombinant psoriasin. We showed that VEGF expression was not induced by hydrogen peroxide, when psoriasin was silenced by shRNA, which led to the hypothesis that psoriasin induces ROS. Indeed, psoriasin was shown to induce ROS in both endothelial and epithelial cells. Moreover, sRAGE inhibited the psoriasin-dependent generation of ROS in endothelial cells. Finally, treatment with antioxidant Bcl-2 protein abolished the effect of psoriasin on endothelial cell proliferation. Our data suggest that psoriasin expression in mammary epithelial cells leads to increased endothelial cell proliferation in a paracrine manner through RAGE. Psoriasin may therefore play a role in breast cancer progression by promoting oxidative stress response and angiogenesis.funding agencies|Swedish Cancer Society||Swedish Psoriasis Association||Assar Gabrielsson Foundation||Welander Foundation||Tore Nilsson Foundation||</p

Psoriasin (S100A7) increases the expression of ROS and VEGF and acts through RAGE to promote endothelial cell proliferation

Psoriasin (S100A7), originally identified in psoriasis, is a calcium-binding protein belonging to the multigenic S100 family. In high-grade ductal carcinoma in situ, psoriasin was identified as one of the most abundant transcripts. We have previously shown that psoriasin was induced by reactive oxygen species (ROS). Moreover, the downregulation of psoriasin by short hairpin RNA (shRNA) led to the reduced expression of vascular endothelial growth factor (VEGF) and inhibited tumor growth in vivo. The aim of the present study was to investigate whether psoriasin could have direct effects on endothelial cells. In this study we demonstrated that psoriasin increased VEGF expression in mammary epithelial cells. The treatment of endothelial cells with recombinant psoriasin increased proliferation comparable to that of recombinant VEGF protein. No change in proliferation was seen when endothelial cells were infected with psoriasin-expressing adenoviruses, suggesting that the proliferative effect of psoriasin was mediated by a specific receptor. Treatment with sRAGE, targeting the receptor for advanced glycation end products (RAGE), thus inhibited endothelial cell proliferation and tube formation enhanced by recombinant psoriasin. We showed that VEGF expression was not induced by hydrogen peroxide, when psoriasin was silenced by shRNA, which led to the hypothesis that psoriasin induces ROS. Indeed, psoriasin was shown to induce ROS in both endothelial and epithelial cells. Moreover, sRAGE inhibited the psoriasin-dependent generation of ROS in endothelial cells. Finally, treatment with antioxidant Bcl-2 protein abolished the effect of psoriasin on endothelial cell proliferation. Our data suggest that psoriasin expression in mammary epithelial cells leads to increased endothelial cell proliferation in a paracrine manner through RAGE. Psoriasin may therefore play a role in breast cancer progression by promoting oxidative stress response and angiogenesis.funding agencies|Swedish Cancer Society||Swedish Psoriasis Association||Assar Gabrielsson Foundation||Welander Foundation||Tore Nilsson Foundation||</p

Psoriasin and MUC1 expression is elevated in CD24⁺ mammary epithelial cells.

<p>MCF10A cells were cultured in confluence for 10 days and separated for CD24⁺ cells, using magnetic activated cell sorting. <b>A</b> Psoriasin expression, analyzed by Western blotting, was confined to the CD24⁺ cell fraction, compared with negative selection and controls. Equal loading was confirmed by GAPDH. The figure illustrates a representative example (n = 3). <b>B</b> Separated CD24⁺ cells showed an increase in the expression of CD24 and a decrease in the expression of CD44, compared with negative selection. The expression of MUC1 was increased in the same level as CD24 expression. The figures illustrate representative examples (n = 4).</p

Endogenous psoriasin causes increased CD24 expression.

<p>MCF10A cells were transfected with a psoriasin-targeting shRNA (Pso-shRNA) or siRNA (Pso-siRNA), and their corresponding controls,(C-shRNA and C-siRNA). Transfected MCF10A cells were cultured in confluence for 10 days or in suspension for 3 days. The expression level of psoriasin was detected by Western blot. CD24 and CD44 expression were measured using flow cytometry. <b>A</b> No induction of psoriasin expression was observed in Pso-shRNA, compared with C-shRNA, in confluence or suspension. <b>B</b> Pso-shRNA showed a decrease in the expression of CD24 and an increase in the expression of CD44, compared with C-shRNA, during confluence and suspension. <b>C</b> The expression of psoriasin in Pso-siRNA was dramatically downregulated, compared with C-siRNA. <b>D</b> Pso-siRNA in suspension culture showed a reduced CD24 expression, compared with C-siRNA. Equal loading was confirmed by GAPDH. The figures illustrate representative examples (n = 3). The data are presented as the mean ± SD of relative expression (n = 3). The p-values (*<0.05) were calculated using the Student's t-test.</p

Confluence- and suspension-cultured mammary epithelial cells demonstrate increased psoriasin and CD24 expression.

<p>MCF10A cells were cultured in confluence for 5 and 10 days or in suspension for 3 days. <b>A</b> Psoriasin expression, analyzed by Western blotting, was induced in MCF10A cells cultured in confluence and suspension, compared with exponentially growing cells. Equal loading was confirmed by GAPDH. The figure illustrates a representative example (n = 3). <b>B</b> In confluence- and suspension-cultured cells, psoriasin and CD24 expression were increased, whereas CD44 expression was decreased compared with exponentially growing cells. The expression level of CD24 and CD44 was measured using flow cytometry and the expression level of psoriasin was quantified from Western blots. The data are presented as the mean ± SD of relative expression (n = 3). The p-values (*<0.05, **<0.01, ***<0.001) were calculated using the Student's t-test.</p

Inhibition of ROS and the NF-κB signaling pathway suppresses psoriasin and CD24 expression.

<p>MCF10A cells were cultured in confluence for 10 days. The inhibition of ROS using NAC (<b>A</b>) and NF-κB using CAPE (<b>B</b>) and dnIKKB (<b>C</b>) led to a significantly reduction in CD24 and psoriasin expression in confluence-cultured MCF10A, measured by flow cytometry and Western blot, respectively. No reduction of CD24 expression was seen by the inhibition of PLC-IP3 using U73122 (<b>D</b>). The reduced CD24 expression by Tyrphostin (<b>E</b>) did not reach statistical significance. The inhibition of PI3-K by Wortmannin (<b>F</b>) showed no decrease in psoriasin or CD24 expression. The inhibitors showed no effect on CD44 expression, except for treatment with NAC (<b>A</b>), which increased the expression of CD44. Western blot inserts illustrate representative examples (n = 4) of the psoriasin expression. Equal loading was confirmed by GAPDH. The data are presented as the mean ± SD of relative expression (n = 4). The p-values (*<0.05, **<0.01, ***<0.001) were calculated using the Student's t-test.</p