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First published online 3 May 2005
doi: 10.1242/jcs.02352

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Expression of truncated latent TGF-ß-binding protein modulates TGF-ß signaling

Roberta Mazzieri^1,*,, Vladimir Jurukovski^1,*,, Hiroto Obata^1,¶, Joanne Sung¹, Alec Platt^1,**, Eric Annes^1,, Nevena Karaman-Jurukovska², Pierre-Emmanuel Gleizes^1, and Daniel B. Rifkin^1,3,¶¶

¹ Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
² Department of Oral Biology and Pathology, State University of New York at Stony Brook, Westchester Hall, Stony Brook, NY 11794-8702, USA
³ Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

View larger version (32K): [in a new window]   Fig. 1. LTBP-1, ECR3E-Myc, ECR3E-1-2-Myc and transgene constructs. (A) LTBP-1 structure and its binding to LAP and TGF-ß through the CR3 region. Ovals represent CR domains, rectangles EGF-like domains; LAP is in black, and mature TGF-ß in orange. (B) The amino acid sequence of the CR3 region of the LTBP-1 (ECR3E-Myc) and the mutations introduced to convert ECR3E-Myc into CR3 that resembles that of LTBP-2 (ECR3E-1-2), which does not bind TGF-ß. The amino acid changes are boxed and the deleted amino acids are labeled with arrows. (C) Transgene products. The two CR3 regions (oval) surrounded by the EGF-like repeats (rectangles) tagged with Myc tag (long rectangle) that were used in making the transgene constructs represented in D. (D) K14 transgene constructs. The keratin 14 (K14) promoter was used to drive the expression of ECR3E-Myc constructs into the basal layer of the epidermis. The human growth hormone 3' untranslated region and poly A (hGH poly A) were added for stabilization of the transcript. Primers used for PCR are represented by arrows and the fragment of the hGH used for Southern blotting indicated by the bold lines.

View larger version (32K): [in a new window]   Fig. 2. Expression of the ECR3E-Myc and ECR3E-1-2-Myc proteins and competition with endogenous LTBP for binding to the small latent complex (SLC) in transfected cells. Western blot analyses of conditioned media of ECR3E-Myc, ECR3E-1-2-Myc or mock transfected HT1080 cells. (A) Reducing conditions and anti-Myc antibody. (B) Nonreducing conditions and anti-LTBP antibody. (C) Nonreducing conditions and anti-LAP antibody.

View larger version (18K): [in a new window]   Fig. 3. Co-immunoprecipitation of ECR3E and TGF-ß activity from primary keratinocytes. Wild-type and transgenic (ECR3E) primary keratinocytes were isolated and grown in low calcium medium. Conditioned medium was collected and used in immunoprecipitation (IP) experiments with either anti-Myc (A14) or anti-LTBP (Ab39) antibodies. Nonimmune rabbit-IgG (RIgG) or rabbit serum (RS) were used as negative controls. Immunoprecipitates were analyzed for TGF-ß activity using the PAI-1-luciferase assay as described in Materials and Methods. PAI-1-luciferase transfected mink lung cells were grown for 16 hours in the presence of heat-activated wild-type (wt) or transgenic (tg) immunoprecipitates in the absence or in the presence of LAP or neutralizing isoform-specific anti-TGF-ß antibodies (anti-TGF-ß1, anti-TGF-ß2 and anti-TGF-ß3).

View larger version (65K): [in a new window]   Fig. 4. Expression of the transgenes in vivo. (A) The Myc-tagged transgenes were detected using anti-Myc antibody in sections of back skin from 9-day-old mice. Both ECR3E-Myc and ECR3E-1-2-Myc transgenic animals showed labeling in the basal layer of the epidermis and the outer root sheet (ORS) of the hair follicle. Control (WT) littermates did not show significant labeling of these regions. (B) Expression of the TGF-ß1 and LTBP-1 visualized using RNA extracted from isolated epidermis or cultured keratinocytes followed by RT-PCR. (C) Wild-type and transgenic (ECR3E-Myc) mice at day 11.

View larger version (85K): [in a new window]   Fig. 5. Histological analyses of the hair cycle. Skin sections from mice stained with hematoxylin and eosin were analyzed at days 0-21. (A) Wild-type (WT) tissues at day 15 displayed hair morphology typical of the late postnatal hair follicle morphogenesis phase, with well-differentiated hair follicles characterized by large oval hair bulbs surrounding the dermal papillae (arrow). At the same day after birth, the ECR3E-Myc transgenic follicles had already entered the catagen phase as evidenced by the regressing follicles with the papilla outside of the hair bulb (arrow). (B) The control ECR3E-1-2-Myc skin had a hair cycle similar to that of the wild-type skin as illustrated by the fact that the hair was in late postnatal hair follicle morphogenesis phase at day 15 and was not distinguishable from wild-type follicles of the same age.

View larger version (61K): [in a new window]   Fig. 6. Detection of phosphorylated Smad2/3 (pSmad2/3) in the skin of wild-type and transgenic animals during the first hair cycle. (A) Immunocytochemistry using pSmad2/3 antibody and Alexafluor visualization of activated Smad2/3 on sections from 9-day-old mice. The positive pSmad2/3 signal stained red. The DAPI-stained nuclei, which were blue, were artificially colored green. Therefore, cells with pSmad2/3 in the nucleus appeared yellow-orange because of the red green overlap. Green arrows indicate pSmad-negative nuclear staining, yellow arrows indicate pSmad-positive nuclear staining. (B) The number of pSmad-positive cells in hair follicles is represented as a percentage of the total number of cells in at least eight fields from each sample of skin from animals 0 to 26 days after birth. The follicles in the skin of ECR3E-Myc animals have higher levels of positive cells compared with the follicles of the wild-type and ECR3E-1-2-Myc.

View larger version (54K): [in a new window]   Fig. 7. Model for latent TGF-ß activation in ECR3E-Myc and ECR3E-1-2-Myc skin. (A) In the presence of wild-type LTBP and/or ECR3E-1-2-Myc, the latent TGF-ß SLC is bound to LTBP and sequestered into the matrix. This prevents latent TGF-ß from interacting with cell-associated activators until the appropriate time. (B) In the situation in which ECR3E-Myc is overproduced, it out-competes LTBP for binding to SLC. The complex of ECR3E-Myc and SLC is not incorporated into the ECM as it is missing the N- and C-terminal matrix binding sites of LTBP. The soluble latent TGF-ß complex, therefore, interacts with cell-associated activators generating active TGF-ß, which causes increased signaling through its receptor and accumulation of nuclear pSmad2/3.