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First published online 12 December 2006
doi: 10.1242/jcs.03317

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Sumoylation dynamics during keratinocyte differentiation

¹ Department of Molecular and Microbial Pathogenesis, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114, USA
² Department of Molecular Genetics and Microbiology, and of Obstetrics and Gynecology, University of New Mexico School of Medicine, 915 Camino de Salud NE, Cancer Research Facility (CRF) 303, Albuquerque, NM 87131, USA

View larger version (40K): [in this window] [in a new window]   Fig. 1. HaCaT cell cultures exhibit phenotypic differences in response to Ca2+ concentration. (A) Phase-contrast microscopy of HaCaT cells maintained in low [Ca2+] medium (0.03 mM Ca2+) or in high [Ca2+] medium (2.8 mM Ca2+). Upon Ca2+ addition to the medium, basal HaCaT cells take 4-7 days to assume a complete differentiated state. Inversely, upon Ca2+ depletion, differentiated HaCaT cells take about 3 weeks to revert to a basal phenotype. (B) RT-PCR analyses of total mRNA harvested from basal (0.03 mM Ca2+) and differentiated HaCaT (2.8 mM Ca2+) cultures. (C) Immunoblot analyses showing expression of keratin 1, involucrin, and -tubulin in total cell extracts harvested from basal (0.03 mM Ca2+) and differentiated (2.8 mM Ca2+) HaCaT cultures.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Components of the sumoylation system are expressed in basal and differentiated HaCaT cells. RNA (A-C) or protein (D,E) were harvested from HaCaT cells maintained in either low [Ca2+] (0.03 mM) or high [Ca2+] (2.8 mM) medium. Harvesting and analysis were performed as described in Materials and Methods. (A) Quantitative RT-PCR of involucrin mRNA levels. (B) RT-PCR analysis of the indicated sumoylation system genes. (C) Quantitative RT-PCR analysis of the relative levels of Ubc9, SAE1, SAE2, SUMO1, SUMO2 and SUMO3 mRNAs. (D) Immunoblots for SAE1, Ubc9 and -tubulin. SAE1 and Ubc9 blots were quantified by densitometry and normalized to -tubulin. The graph shows the relative protein levels for HaCaT cells maintained in low and high [Ca2+] medium, and the lower panels show representative SAE1 and Ubc9 immunoblots with their corresponding -tubulin controls. (E) Anti-SUMO immunoblot of total cell extracts from HaCaT cells maintained in either low or high [Ca2+] medium. Protein concentrations in the extracts were normalized to -tubulin, and the samples were resolved by 10% SDS-PAGE. Arrows indicate sumoylated proteins whose levels appear higher in the high [Ca2+] culture. Quantitative results in A, C and D are the average of at least three independent experiments.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Profile of SUMO and Ubc9 expression in organotypic cultures of human foreskin keratinocytes. Tissue sections were processed and evaluated for protein expression using antibodies to Ubc9 (A,B), SUMO (B,D), or human keratin 1 (E). For samples B and D, the respective purified proteins (25 µg Ubc9 or 7 µg SUMO1) were included in the primary antibody incubation step to block detection of the corresponding antigens. (F) Negative control where the primary antibody was absent. All sections were counterstained with hematoxylin. Arrows indicate cells expressing high levels of Ubc9. Arrowheads indicate cells in the basal layer exhibiting low levels of Ubc9 expression.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Keratinocyte differentiation is accompanied by transcription upregulation of the sumoylation system. HaCaT cells maintained in low [Ca2+] medium (Time 0) were induced to differentiate by replacing the medium with high [Ca2+] medium. RNA was extracted at various times post Ca2+ induction as indicated. The extracted RNA was analyzed for expression of differentiation marker genes (A,B) or sumoylation system genes (C-E), and the mRNAs were detected either by quantitative RT-PCR (A,C,E) or RT-PCR (B,D). For the quantitative RT-PCR, the 0 hour time samples were set to a value of 1, and the values at other time points are relative to the 0 hour value. For the RT-PCR, 18S rRNA was used as the internal standard. The quantitative results in A,C and E were the average of at least three independent experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Keratinocyte differentiation is accompanied by a transient upregulation of several sumoylation system proteins. Parallel HaCaT cell cultures were maintained either in low [Ca2+] medium (Uninduced) or switched from low [Ca2+] to high [Ca2+] medium (Induced). At the indicated times post Ca2+ induction, protein extracts were prepared from the paired cultures. The extracts were assayed for expression of K1 (A), Ubc9 (B), SAE1 (B), and SENP1 (B) by immunoblotting with specific antibodies. The induced and uninduced cell extracts were also evaluated for -tubulin expression, and the immunoblots for -tubulin are duplicated in each panel of B for comparative purposes. (C) The immunoblots in B were quantified by densitometry, and the values for Ubc9, SAE1, and SENP1 were normalized against -tubulin. The 0 hour time values for both the induced and uninduced samples were set to 1, and the values for the later time points are relative to the 0 hour time. The quantitative results in C are the average of at least three independent experiments.

View larger version (83K): [in this window] [in a new window]   Fig. 6. The pattern of SUMO-conjugated substrates changes during HaCaT cell differentiation. Total cell extracts were prepared at the indicated times post Ca2+ induction and analyzed by immunoblotting using antibodies to SUMO1 (upper panel), -tubulin (middle panel) or keratin 1 (lower panel). Protein concentrations in the samples were equalized based on the -tubulin levels. The positions of molecular size markers are shown on the right. Arrows and arrowheads indicate bands whose quantity increased or decreased, respectively, during the time period examined.

View larger version (67K): [in this window] [in a new window]   Fig. 7. Inhibition of sumoylation perturbs normal HaCaT cell differentiation. (A) HaCaT cells undergoing Ca2+-induced differentiation exhibit abnormal morphology when expressing Gam1 protein. Shown are phase-contrast and fluorescence microscopy images of HaCaT cell cultures at 24-hour intervals. HaCaT cells were infected with Ad-Gam1/Ad-GFP (upper panel; MOI 150/150) or Ad-GFP alone (middle and bottom panels; MOI 300) and induced to differentiate by switching to high [Ca2+] medium. For the bottom panel, 100 mM TSA (an HDAC inhibitor) was added 24 hours post infection and induction and was re-added in fresh medium at 48-hour intervals. (B) Immunoblot analysis for Ubc9, Gam1, and -tubulin in infected HaCaT cells. Cells were infected with the Ad-Gam1 (MOI 300), Ad-Gam1/Ad-GFP (MOI 150/150), or Ad-GFP (MOI 300) and induced to differentiate as in A. Cell extracts were prepared at the indicated times and immunoblotted. (C) Immunoblot analysis for keratin 1 and -tubulin. Cells were infected with the indicated adenovirus constructs at an MOI of 300. Cell extracts were prepared and analyzed as in B.

View larger version (12K): [in this window] [in a new window]   Fig. 8. Model for differentiation-dependent changes in the sumoylation system. The model proposes a negative-feedback mechanism to explain the transient increase in sumoylation observed during HaCaT cell differentiation. Initially, a pool of TFs, including Sp1, C/EBP and AP1, which are known to be directly stimulated by Ca2+-induced differentiation, cause upregulation of the sumoylation system. As sumoylation activity increases, these TFs are in turn modified by SUMO conjugation, which decreases their transcriptional activity and leads to a decline in expression of the sumoylation pathway genes. A second feature of the model is that increased sumoylation is an active contributor to the differentiation process, through SUMO conjugation to downstream effectors of the differentiation signals. Additional details are provided in the text.

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