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First published online 19 April 2005
doi: 10.1242/jcs.02303

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Increased keratinocyte proliferation by JUN-dependent expression of PTN and SDF-1 in fibroblasts

¹ Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
² Division of Differentiation and Carcinogenesis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
³ Institute of Cell Biology, ETH Zürich, Hoenggerberg, 8093 Zürich, Switzerland

View larger version (28K): [in a new window]   Fig. 1. Expression of PTN and SDF-1 is JUN dependent. (A) Expression profiling of wild-type versus Jun–/– mouse embryonic fibroblasts revealed Ptn and Sdf-1 to be activated by JUN. Enhanced transcript levels in wild-type cells (black bars) are indicated as fold difference compared to the expression in Jun–/– cells (white bars), which was set as 1. Kgf, representing a known direct JUN target, the JUN-repressed disabled homolog 2 (Dab2) and the JUN-independent genes lipocalin (Lcn2) and CD24a antigen (cd24a) were included as controls. (B) Validation of the microarray results by semi-quantitative RT-PCR on cDNA derived from untreated and IL-1-stimulated fibroblast monocultures. Expression of the JUN-activated cytokines Ptn, Sdf-1 and Kgf was impaired in fibroblasts lacking JUN activity, whereas Cxcr4, Lcn2, Dab2 and Cd24a levels were not reduced. ß-tubulin 1 (Tubb1) was used for standardization.

View larger version (63K): [in a new window]   Fig. 2. PTN and SDF-1 are expressed by dermal fibroblasts in vivo. (A) Dermis and epidermis of mouse back skin were dissected and semi-quantitative RT-PCR was performed to reveal the expression of Ptn and SDF-1 in the dermal compartment. Keratin 10 (K10) and procollagen type I, 2 (Col1a2) were used as controls for the separation. The cDNA was standardized to Hprt levels. (B) In situ hybridization using DIG-labelled riboprobes for Ptn and Sdf-1 confirmed dermal fibroblasts as a source of cytokine expression. Hybridization was visualized using diaminobenzidine (DAB, reddish-brown signal) and nuclei were counterstained with Hematoxylin.

View larger version (56K): [in a new window]   Fig. 3. SDF-1 and PTN stimulate proliferation of primary keratinocytes in vitro. (A) Heterologous feeder-layer co-cultures grown in the presence or absence of recombinant human SDF-1 (50 ng/ml) or PTN (25 ng/ml) were analysed by phase contrast microscopy (left column) and immunofluorescence (right columns). Individual keratinocyte islands are outlined with dashed lines in the phase contrast images. Differentiation and proliferation of primary keratinocytes was visualized by staining for keratin 10 (early differentiation), involucrin and MIB1 (proliferation). (B) Quantification of the mitogenic effect of PTN and SDF-1 on primary keratinocytes. MIB1-positive cells of at least 10 colonies were counted and plotted with the total number of cells per keratinocyte colony (shaded bars).

View larger version (62K): [in a new window]   Fig. 4. Neutralization of SDF-1 activity impairs keratinocyte proliferation (A) Phase contrast microscopy demonstrating the inhibitory effect of neutralizing antibodies against either SDF-1 (10 µg/ml) or its receptor CXCR4 (5 µg/ml) on keratinocyte growth. Individual islands are outlined with dashed lines. (B) Proliferating cells and colony size were quantified as in Fig. 3B.

View larger version (37K): [in a new window]   Fig. 5. Regulation of PTN and SDF-1 in wounds and co-cultured fibroblasts. (A) Real-time PCR and (B) semi-quantitative RT-PCR was performed on cDNA from full-thickness excisional mouse dorsal skin wounds prepared at the indicated times after injury. (A) Changes in mRNA levels during the wound healing process are plotted relative to the expression in unwounded skin (0 hours, set as 1). Average values of three independent measurements are shown. (B) Expression analysis on RNA from an independent wounding experiment. Essentially identical results were obtained in a third independent RNA analysis of expression kinetics during wound healing (data not shown). Gro1 (A,B) and Kgf (B), both known to be induced by wounding, were included as positive controls. (C) Expression of Ptn, Sdf-1 and Cxcr4 in fibroblast monocultures or co-cultures with primary human normal epidermal keratinocytes (NEKs). Ptn and Cxcr4 were upregulated, whereas Sdf-1 transcript levels decreased in the presence of co-cultured keratinocytes.

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