Kamalakar C. Nerusu

Regulation of E-cadherin and β-catenin by Ca2+ in colon carcinoma is dependent on calcium-sensing receptor expression and function

An siRNA directed against the extracellular calcium‐sensing receptor (CaSR) was used to down‐regulate this protein in CBS colon carcinoma cells. In additional studies, we utilized a variant of the parental CBS line that demonstrates CaSR expression but does not upregulate this protein in response to extracellular Ca2+. In neither the siRNA‐transfected cells nor the Ca2+‐nonresponsive variant cells did inclusion of Ca2+ in the culture medium inhibit proliferation or induce morphological alterations. Extracellular Ca2+ also failed to induce E‐cadherin production or a shift in β‐catenin from the cytoplasm to the cell membrane. In mock‐transfected cells and in a Ca2+‐responsive variant line derived from the same parental CBS cells, Ca2+ treatment resulted in growth‐reduction. This was accompanied by increased E‐cadherin production and a shift in β‐catenin distribution from the cytoplasm to the cell membrane. Additionally, down‐regulation of c‐myc and cyclin D1 expression was observed in mock‐transfected cells and in the Ca2+‐responsive variant line (along with reduced T cell factor transcriptional activation). Neither c‐myc nor cyclin D1 was significantly down‐regulated in the siRNA‐transfected cells or in the Ca2+‐nonresponsive variant cells upon Ca2+ stimulation. In histological sections of human colon carcinoma CaSR was significantly reduced as compared to the level in normal colonic crypt epithelial cells. Where CaSR expression was high, strong surface staining for E‐cadherin and β‐catenin was observed. Where CaSR expression was reduced, β‐catenin surface expression was likewise reduced.

Amphiregulin and Epidermal Hyperplasia: Amphiregulin Is Required to Maintain the Psoriatic Phenotype of Human Skin Grafts on Severe Combined Immunodeficient Mice

Overexpression of amphiregulin has been shown to induce psoriasiform changes in the skin of transgenic mice shortly after birth. Therefore, amphiregulin has been suggested as a target for anti-psoriatic therapy. To test this theory, a humanized monoclonal antibody capable of neutralizing human amphiregulin was examined for anti-proliferative effects in the human skin-severe combined immunodeficient (SCID) mouse transplant model. The anti-amphiregulin antibody reduced epidermal thickness of transplanted psoriatic skin and also inhibited the hyperplastic response that developed in nonpsoriatic skin after transplantation. The same antibody also suppressed keratinocyte proliferation in monolayer culture in a dose-dependent manner. Under the same conditions in which keratinocyte proliferation was inhibited, the antibody had little effect on proliferation of human dermal fibroblasts and no effect on type I procollagen production by these cells. Taken together, these data indicate an important role for amphiregulin in psoriatic hyperplasia and suggest that inhibition of amphiregulin activity could be an efficacious therapeutic strategy for psoriasis. These data also suggest that the hyperplastic response occurring in nonpsoriatic human skin on transplantation to the SCID mouse is mediated, in large part, by amphiregulin.

Role of Metalloelastase in a Model of Allergic Lung Responses Induced by Cockroach Allergen

Our laboratory and others have shown an important role of metalloelastase (MMP-12) in the pathogenesis of acute and chronic lung injury. Because chronic asthma is characterized by airway inflammation and alterations in the airway extracellular matrix, we explored the role of metalloelastase in a model of allergic airway inflammation induced by cockroach antigen (CRA). Using MMP-12-deficient mice we found a significant reduction in CRA-induced inflammatory injury, as evidenced by fewer peribronchial leukocytes, significantly less protein in the bronchoalveolar lavage (BAL) fluid, and a significant reduction in the number of infiltrating neutrophils, eosinophils, and macrophages, relative to wild-type mice. Although we did not find a significant reduction in the number of T cells in the injured MMP-12-deficient animals as compared to controls, levels of the chemotactic factors interleukin-5, macrophage inflammatory protein-1 alpha, monocyte chemoattractant protein-1, thymus activation regulated chemokine, and the proinflammatory cytokine tumor necrosis factor-alpha were significantly reduced in the bronchoalveolar lavage fluid of CRA-challenged MMP-12-deficient mice, relative to CRA-challenged control animals. These studies indicate that MMP-12 plays an important proinflammatory role in the development of allergic inflammation in the CRA model. Alterations in the levels of chemotactic factors and other proinflammatory cytokines in the MMP-12-deficient mice may underlie the decrease in leukocyte recruitment into inflamed lungs.

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423), a Benzodiazepine, Suppresses Keratinocyte Proliferation and Has Antipsoriatic Activity in the Human Skin-Severe, Combined Immunodeficient Mouse Transplant Model

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a benzodiazepine that has cytotoxic and cytostatic activity against a variety of cells in vivo and in vitro. In the present study, we demonstrate that Bz-423 (formulated for topical delivery) reduces epidermal hyperplasia in human psoriatic skin after transplantation to severe, combined immunodeficient (scid) mice. Bz-423 also suppresses the hyperplasia that develops in nonpsoriatic human skin as a consequence of transplantation to scid mice. Proliferation of human epidermal keratinocytes in monolayer culture was suppressed by Bz-423 at concentrations of 0.5 to 2.0 μM (noncytotoxic concentrations). Keratinocyte growth inhibition was accompanied by increased oxidant generation in Bz-423-treated cells, and treatment with vitamin E along with Bz-423 reversed the growth inhibition. Growth inhibition was accompanied by a redistribution of β-catenin from a cytoplasmic pool to the cell membrane and by reduced levels of c-myc and cyclin D1 (two molecules associated with Wnt pathway signaling). Several analogs of Bz-423 were examined for antiproliferative activity against human epidermal keratinocytes and human dermal fibroblasts in monolayer culture. Each of the analogs tested suppressed growth of both cell types, but in all cases, keratinocytes were more sensitive than fibroblasts. Two of the compounds were found to suppress epidermal hyperplasia induced with all-trans retinoic acid in organ cultures of human skin. Taken together, these data show that Bz-423 and certain analogs produce biological responses in skin cells in vitro and in vivo that are consistent with therapeutic goals for treating psoriasis or epidermal hyperplasia resulting from other causes.

Vascular expression of matrix metalloproteinase-13 (collagenase-3) in basal cell carcinoma

Matrix metalloproteinase-13 (MMP-13; collagenase-3) was detected in the vasculature from 17 of 20 human basal cell carcinomas as assessed by immunohistology immediately after surgery. In contrast, MMP-1 (interstitial collagenase) was detected in the vasculature of only two of the same specimens. MMP-13 reactivity was also observed in the capillaries of normal human skin taken from the wound margin. Human dermal microvascular endothelial cells as well as human umbilical vein endothelial cells were isolated in culture and examined for MMP-13 expression. By reverse transcription-polymerase chain reaction and Southern blotting, an MMP-13 transcript was detected in unstimulated endothelial cells. The transcript was upregulated in cells treated with 50 nM phorbol myristate acetate (PMA). Western blotting demonstrated the presence of an anti-MMP-13 - immunoreactive protein in culture fluid from both cell sources. Immunoreactivity was stronger in culture fluid from cells treated with interleukin-1alpha (IL-1alpha) than in culture fluid from control cells. Tumor necrosis factor-alpha (TNF-alpha) and PMA also upregulated MMP-13 expression but these agents were not as effective as IL-1alpha. Additionally, reactivity was greater in culture fluid from endothelial cells grown on three-dimensional lattices of polymerized type I collagen than on dried collagen films. These data indicate that endothelial cells in the skin are a source of MMP-13 and that enzyme expression is upregulated under conditions that promote endothelial cell growth and vascular differentiation.

MDI 301, a nonirritating retinoid, improves abrasion wound healing in damaged/atrophic skin

MDI 301 is a picolinic acid‐substituted ester of 9‐cis retinoic acid. It has been shown in the past that MDI 301 increases epidermal thickness, decreases matrix metalloproteinase (MMP) activity, and increases procollagen synthesis in organ‐cultured human skin. Unlike all‐trans retinoic acid (RA), MDI 301 does not induce expression of proinflammatory cytokines or induce expression of leukocyte adhesion molecules in human skin. In the present study we examined topical MDI 301 treatment for ability to improve the structure and function of skin in three models of skin damage in rodents and for ability to improve abrasion wound healing in these models. MDI 301 was applied daily to the skin of rats treated with the potent corticosteroid, clobetasol propionate, to the skin of diabetic rats (8 weeks posttreatment with streptozotocin) and to the skin of aged (14–16‐month‐old) rats. In all three models, subsequently induced abrasion wounds healed more rapidly in the retinoid‐treated animals than in vehicle‐treated controls. Immediately after complete wound closure, tissue from the wound site (as well as from a control site) was put into organ culture and maintained for 3 days. At the end of the incubation period, culture fluids were assessed for soluble type I collagen and for MMPs‐2 and ‐9. In all three models, the level of type I collagen was increased and MMP levels were decreased by MDI 301. In all three models, skin irritation during the retinoid‐treatment phase was virtually nonexistent.