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First published online 31 July 2007
doi: 10.1242/jcs.03480

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Reduced migration, altered matrix and enhanced TGF1 signaling are signatures of mouse keratinocytes lacking Sdc1

Mary Ann Stepp^1,2,*, Yueyuan Liu¹, Sonali Pal-Ghosh¹, Rosalyn A. Jurjus¹, Gauri Tadvalkar¹, Adith Sekaran¹, Kristen LoSicco¹, Li Jiang³, Melinda Larsen^4,, Luowei Li⁵ and Stuart H. Yuspa⁵

¹ Department of Anatomy and Cell Biology, George Washington University Medical School, Washington, DC 20037, USA
² Department of Ophthalmology, George Washington University Medical School, Washington, DC 20037, USA
³ Institute for Biomedical Engineering, School of Engineering and Applied Science, George Washington University, Washington, DC 20037, USA
⁴ National Institute of Dental and Craniofacial Research/Laboratory of Cellular and Developmental Biology, National Institutes of Health, Bethesda, MD 20892, USA
⁵ National Cancer Institute/Laboratory of Cancer Biology and Genetics, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (71K): [in this window] [in a new window]   Fig. 1. Although loss of Sdc1 alters keratinocyte growth characteristics, Sdc1-null keratinocytes retain their epithelial morphology and keratin expression. (A,B) Equivalent numbers of primary wt (+/+) and Sdc1-null (–/–) keratinocytes were plated out and grown for the times indicated; in A keratinocytes are viewed with phase-contrast optics at 3 days after being placed in culture, and in B the numbers of adherent primary +/+ and –/– keratinocytes were counted at the indicated days. Data are plotted as the mean ± s.e.m.; *, significantly more Sdc1-null keratinocytes per dish than wt keratinocytes at days 3 and 10. Bar in A, 10 µm. (C) Studies using [3H]thymidine showed that at day 7, Sdc1-null keratinocytes proliferated more than wt keratinocytes, even after controlling for differences in keratinocyte density. (D) Triple-labeling using FITC-labeled phalloidin (green) for F-actin, K14 (red) for intermediate filament protein keratin 14, and DAPI (blue) for nuclei in +/+ keratinocytes (a-d) and on –/– keratinocytes (e-h). The localization of K14 appears similar in wt and Sdc1-null keratinocytes. Sdc1-null keratinocytes show thicker cortical actin filament bundles that localized prominently at keratinocyte peripheries compared with wt keratinocytes. *, regions shown magnified in d and h to better emphasize the actin cortical filaments. Bar in D, 4 µm (a-c and e-g) and 1.3 µm (d and h).

View larger version (37K): [in this window] [in a new window]   Fig. 2. Keratinocyte adhesion and time-lapse migration studies show that Sdc1-null keratinocytes adhere better and migrate poorly compared with wt keratinocytes, but only when migrating on matrix they produce themselves. (A) Cell adhesion studies were performed on equal numbers of wt and Sdc1-null keratinocytes, which were allowed to adhere on wells coated with fibronectin (FN), vitronectin (VN), laminin-111 (LN-111), collagen I (CN-I) and collagen IV (CN-IV). Note that the Sdc1-null keratinocytes are significantly more adherent than the wt keratinocytes to all ECM molecules tested. (B,C) Cell migration was assessed in time-lapse experiments in wt and Sdc1-null keratinocytes 5 days after initial plating. For details on the quantification, see Materials and Methods. Velocity measurements for wt and Sdc1-null keratinocytes are presented in B. *P<0.05. Typical tracks (red) of 15 keratinocytes are shown superimposed over final relief-contrast images of wt and Sdc1-null keratinocytes in C. Bar, 10 µm. (D) Velocities of wt and Sdc1-null keratinocytes were compared after replating onto FNCNI matrix and onto LN-332 as well as onto matrices deposited by each keratinocyte genotype. Data show that after replating, Sdc1-null keratinocytes migrated more slowly than wt keratinocytes but only when replated on the Sdc1-null keratinocyte matrix.

View larger version (91K): [in this window] [in a new window]   Fig. 3. Sdc1-null keratinocytes produce a matrix distinct from that made by wt keratinocytes. (A) Sirius Red dye binding assay to measure collagen accumulation in the wt and Sdc1-null cultures shows that the day after the keratinocytes had been tracked, Sdc1-null keratinocyte cultures had accumulated significantly less collagen per keratinocyte compared with wt keratinocytes. (B) Matrix preparations identical to those used in the experiments described in Fig. 2D were extracted as described, normalized and run on 4-20% gels that were silver-stained. High-molecular-mass proteins accumulated in the matrices, but there were no major differences between the amount of high-molecular-mass matrix deposited by the wt and Sdc1-null keratinocytes. The lower molecular mass bands are keratins, which stick non-specifically to the matrix after keratinocytes are lysed. The control extract shows those proteins deposited on wells which remain after ammonium hydroxide treatment of wells that had been coated with FN-CNI and fed serum-containing medium. (C) Immunoblots of the same matrix preparations shown in B normalized by cell count and probed for LN-332 using the J18 antibody showed similar patterns for LN-332 unprocessed (unp) and processed (prc) 332 chains for matrices deposited by wt and Sdc1-null keratinocytes. (D) Immunofluorescence microscopy using the J18 antibody on matrix preparations from cultures of wt and Sdc1-null keratinocytes. (a,d) 20x images taken of wt and Sdc1-null keratinocytes; asterisks indicate elongated clear areas lacking LN-332 surrounded by areas positive for LN-332. In b and e, asterisks indicate regions shown at higher resolution. (c and f) Additional high-resolution images of wt and Sdc1-null keratinocyte matrices, respectively. Arrows in b,c,e,f indicate ordered streaks that are more prominent in Sdc1-null matrix than in wt matrix; arrowheads indicate amorphous cloud-like staining present in wt matrix but largely absent in Sdc1-null keratinocytes. Bar, 6 µm for a,d, 2 µm for b,c,e,f.

View larger version (70K): [in this window] [in a new window]   Fig. 4. The velocity of Sdc1-null keratinocytes can be restored to that of wt keratinocytes by addition of function-blocking antibodies against 4 or v integrins. (A) Time-lapse microscopy analyses were repeated on day 3 wt and Sdc1-null keratinocytes using integrin function-blocking antibodies at concentrations of 25 µg/ml added to serum-containing medium. Data are expressed as fold changes in velocity compared with untreated wt keratinocytes to facilitate comparisons between experiments. Control studies were performed using isotype specific antibodies. Note that the 1 integrin antagonist (9EG7) inhibited wt keratinocyte migration rates significantly by 60% whereas it inhibited Sdc1-null keratinocyte migration by only 30% compared with untreated Sdc1-null keratinocytes, a difference which was not significant. By contrast, antagonizing either 64 integrin using GoH3 or all v integrins using RMV-7 allowed Sdc1-null keratinocytes to migrate at rates similar to those of wt keratinocytes. (B) 4 integrin and LN-332 were simultaneously localized in day 3 wt and Sdc1-null keratinocytes. a,b and e,f show double-staining of actively migrating wt and Sdc1-null keratinocytes, respectively, whereas c,d and g,h show more stationary keratinocytes lacking LN-332 trails. Note the closer association between 4 integrin and LN-332 at the edges of the migrating Sdc1-null keratinocytes in e and f (white arrows) compared with the wt keratinocytes. Bar, 5 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 5. Disruption and activation of TGF1 signaling has distinct effects on the migration rates of wt and Sdc1-null keratinocytes. (A) wt and Sdc1-null keratinocytes were grown for 3 days, after which keratinocytes were treated with a TGF1 neutralizing antibody overnight and then tracked by time-lapse microscopy the next day. Data show that neutralizing TGF1 reduced wt keratinocyte migration rates by over 50% but inhibited Sdc1-null keratinocyte migration by less than 30% compared with untreated or control IgG treated Sdc1-null keratinocytes. (B) wt and Sdc1-null keratinocytes were grown for 3 days, after which keratinocytes were treated with 0.25 or 2.5 ng/ml TGF1 overnight and then tracked the next day. The migration rates of the Sdc1-null keratinocytes were restored to those of wt keratinocytes after treatment with 0.25 ng/ml TGF1. Further, the Sdc1-null keratinocytes migrated significantly faster than the wt keratinocytes when given 2.5 ng/ml TGF1. Whereas lower concentration of TGF1 stimulated wt keratinocyte migration rates, higher concentration did not. *P<0.05; grey line highlights values above untreated wt controls. (C) Localization of LN-332 and 4 integrin in migrating wt and Sdc1-null keratinocytes 24 hours after treatment of keratinocytes with either TGF1-neutralizing antibody (a-d) or with 0.25 ng/ml of TGF1 (e-f). Bar, 5 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Sdc1-null keratinocytes have increased expression of several different integrins on their surface but, unlike wt keratinocytes, they do not increase their integrin surface expression in response to TGF1. (A) Flow cytometry analysis on unfixed isolated wt and Sdc1-null keratinocytes revealed increased surface expression of 1 and 4 integrins, as determined using Student's t-test (P< 0.05). (B) Tests of biotinylation efficiency show that the amount of biotin incorporated per ng total protein for wt and Sdc1-null keratinocytes is similar, as is the overall profile of biotinylated proteins. (C,D) Biochemical analyses of surface integrins using biotinylation and immunoprecipitation reveals elevated levels of several integrins in untreated Sdc1-null keratinocytes, excluding 3 integrin; the increase seen for 2 integrin was not significant. Treating wt and Sdc1-null keratinocytes with 0.25 ng/ml TGF1 for 24 hours significantly increased the expression in wt keratinocytes of all the integrins tested, excluding 31, but had no significant effect on integrin surface expression by the Sdc1-null keratinocytes. Numbers in C represent fold increase in surface integrins in the untreated and TGF1-treated Sdc1-null keratinocytes relative to wt keratinocytes.

View larger version (39K): [in this window] [in a new window]   Fig. 7. TGF1-mediated signaling is altered in Sdc1-null keratinocytes. (A) Addition of increasing concentrations of TGF1 to wt and Sdc1-null keratinocytes inhibits keratinocyte proliferation with both genotypes showing identical responses. (B) A dual reporter assay was used to determine the fold induction of transcription of a TGF1-induced promoter (Smad4) compared with a control promoter (transketolase) 20 hours after wt and Sdc1-null keratinocytes were treated with 2 ng TGF1, as described in Materials and Methods. Note that the baseline of TGF1-mediated gene transcription for untreated keratinocytes was significantly higher for Sdc1-null keratinocytes, and that Sdc1-null keratinocytes had higher TGF1-induced gene expression at all concentrations of TGF1 tested; at 20 hours, these increases were significant for 250 and 500 ng TGF1. (C) Smad2 phosphorylation was measured directly in wt and Sdc1-null keratinocytes before and after TGF1 addition. In response to TGF1, wt keratinocytes showed an increase in P-Smad2, whereas Sdc1-null keratinocytes did not. (D) Amount of total TGF1 secreted into conditioned media was assessed via ELISA assay at the times indicated and data expressed as picogram (pg) per 106 cells. (E) The amount of active TGF1 secreted into conditioned medium as well as in cell extracts was assessed using a standard mink lung epithelial reporter cell assay as described (see Materials and Methods); data are expressed in relative luciferase units.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Cartoon highlighting results from the studies using integrin-function-blocking antibody. PM, plasma membrane; N,nucleus; v, integrin heterodimers that contain the v subunit, are expressed in keratinocytes and include integrins v5, v6 and v8; 1, integrin heterodimers that contain the 1 subunit, are expressed in keratinocytes and include integrins 21, 31 and 51 (see supplementary material Fig. S1 for expression profiles of total keratinocyte integrins and Fig. 6B for surface integrins).

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