The calreticulin-binding sequence of thrombospondin 1 regulates collagen expression and organization during tissue remodeling

Abstract

Amino acids 17-35 of the thrombospondin1 (TSP1) N-terminal domain (NTD) bind cell surface calreticulin to signal focal adhesion disassembly, cell migration, and anoikis resistance in vitro. However, the in vivo relevance of this signaling pathway has not been previously determined. We engineered local in vivo expression of the TSP1 calreticulin-binding sequence to determine the role of TSP1 in tissue remodeling. Surgical sponges impregnated with a plasmid encoding the secreted calreticulin-binding sequence [NTD (1-35)-EGFP] or a control sequence [mod NTD (1-35)-EGFP] tagged with enhanced green fluorescent protein were implanted subcutaneously in mice. Sponges expressing NTD (1-35)-EFGP formed a highly organized capsule despite no differences in cellular composition, suggesting stimulation of collagen deposition by the calreticulin-binding sequence of TSP1. TSP1, recombinant NTD, or a peptide of the TSP1 calreticulin-binding sequence (hep I) increased both collagen expression and matrix deposition by fibroblasts in vitro. TSP1 stimulation of collagen was inhibited by a peptide that blocks TSP1 binding to calreticulin, demonstrating the requirement for cell surface calreticulin. Collagen stimulation was independent of TGF-β activity and Smad phosphorylation but was blocked by an Akt inhibitor, suggesting that signaling through the Akt pathway is important for regulation of collagen through TSP1 binding to calreticulin. These studies identify a novel function for the NTD of TSP1 as a mediator of collagen expression and deposition during tissue remodeling.

Figure 1

Illustration of TSP1-derived molecules used in vivo and in vitro. Platelet-derived trimeric human thrombospondin-1 (TSP1) (aa 1-1152); recombinant TSP1 N-terminal domain trimer (NoC1; aa 1-356); synthetic CRT-binding TSP1 peptide (hep I; aa 17-35); secreted CRT-binding domain EGFP construct [NTD (1-35)-EGFP; TSP 1 aa1-35] and secreted nonfunctional mutated construct [mod NTD (1-35)-EGFP]; and nonsecreted EGFP parent vector. S indicates signal sequence; EGFP, enhanced green fluorescent protein; TSR, type 1 thrombospondin repeat; 2, type 2 thrombospondin repeat.

Figure 2

In vitro, NTD (1-35)-EGFP and mod NTD (1-35)-EGFP are functional substitutes for the synthetic peptides. A: EGFP fusion proteins were detected in the conditioned media of MEFs cells transfected with NTD (1-35)-EGFP or mod NTD (1-35)-EGFP. EGFP-containing proteins were isolated from conditioned media of transfected [EGFP, NTD (1-35)-EGFP or mod NTD (1-35)-EGFP] or control cells (not transfected) 72 hours after transfection by nucleoporation. EGFP containing proteins were isolated by pull-down using mouse anti-GFP coupled beads. Bound proteins were eluted in Laemmli buffer, separated by SDS-PAGE, transferred to nitrocellulose, and then immunoblotted for GFP. B–D: The presence of focal adhesions was assessed by interference reflection microscopy. At least 300 cells were counted per coverslip. Cells with more than five focal adhesions per cell were scored as positive. B: BAECs responded to NTD (1-35)-EGFP in a dose-dependent manner. Conditioned medium from NTD (1-35)-EGFP–transfected cells was diluted 1:10 with conditioned medium from mock-transfected cells. NTD (1-35)-EGFP–conditioned medium (squares), pEGFP-N1-transfected cell conditioned media (circles), and mock-transfected medium (triangles). hep I peptide (10 nmol/L) was used as a positive control for focal adhesion disassembly (dash-dot line). C: Wild-type and CRT (−/−) MEFs were transfected with NTD (1-35)-EGFP constructs. Twenty-four hours after plating, cells were analyzed by interference reflection microscopy for the presence of focal adhesions. Results are expressed as the mean number of focal adhesion positive cells ± SD; n = 3 replicates with P values calculated by two-parameter unpaired Student's t-test. D: NTD (1-35)-EGFP-secreted protein was active in the presence of serum. MEFs were transfected and grown for 24 hours in medium containing 10% FBS. Transfection efficiencies were measured and then transfected cell populations were mixed with mock-transfected cells to normalize the percentage of construct-expressing cells to 10% of each coculture population. Results are the mean percentage of focal adhesion positive cells ± SD; n for each group is indicated on figure. *P < 0.05.

Figure 3

EGFP expression is detected in sponges from days 5–14. A: At day 5, EGFP was detected in vivo at sponge implantation sites by fluorescent imaging. Far left panel shows sponges without fluorescent image overlay. Red arrows: implanted sponges, perimeter of left sponge denoted by a dashed line. Autofluorescence was observed where tissue glue still adheres to the healing incision (collagen and EGFP). Pseudocolor indicates relative intensity of EGFP fluorescence at 515 nm. B: EGFP was detected in the wound fluid from sponges containing NTD (1-35)-EGFP and mod NTD (1-35)-EGFP constructs at day seven. Results are mean molar concentration of EGFP in fluid calculated from an EGFP standard curve of fluorescence with excitation at 485 nm and emission at 530 nm ± SD; n = 6 NTD (1-35)-EGFP sponges and n = 4 modNTD (1-35)-EGFP sponges C: Immunohistochemistry of EGFP expression in sponges at day 14. Paraffin sections of sponges show cells expressing EGFP (arrows). C indicates capsule; S, sponge; GAM, gene-activated matrix infused into sponges. Asterisk indicates the same location at different magnifications. Scale bar = 25 μm.

Figure 4

Collagen capsule formation is increased in NTD (1-35)-EGFP–expressing sponges at days 7 and 14. Sections of representative sponges harvested at days 7 and 14 were stained with Masson's trichrome. Collagen stained blue, muscle and red blood cells stained red, and cell nuclei stained brown. PVA sponge material stained brown/blue at day 7 and blue at day 14. Bracket denotes pericapsular region of the sponge. Plasmid infused collagen (GAM) could be seen between the sponge material (S). Thick collagen capsules (COL) were observed at the perimeter of the sponges beneath the panniculus carnosus dermal muscle layer (PC) in NTD (1-35)-EGFP but not in control plasmid-expressing sponges. Scale bar = 50 μm.

Figure 5

Collagenous ECM capsule density is increased in NTD (1-35)-EGFP sponges. A: Diagram of capsule quantification methodology. Images were captured along the entire pericapsular area of the sponge (left). DM indicates dermal muscle; oc, organized capsule; S, sponge material. The entire capsule region was selected (center) with the area containing organized collagen quantified based on the intensity of blue staining in Masson's trichrome–stained sections (right) using MetaMorph software and expressed as the percentage of the total capsular region that was designated as thresholded by these criteria. B: Total capsular area from sponges harvested at day 7 (filled symbols) and day 14 (open symbols). EGFP (circle), mod NTD (1-35)-EGFP (squares), NTD (1-35)-EGFP (triangles), and collagen only (diamonds). C: Collagen organization is expressed as the percentage of the total area defined by the threshold parameters. P values were calculated using the unpaired two-parameter Student's t-test.

Figure 6

Myofibroblasts are associated with the collagen capsule and granulation tissue in NTD (1-35) and mod NTD (1-35) treatment groups at day seven. Top two panels: Masson's trichrome stain. Bottom two panels: Immunohistochemical staining for α-SMA. Images are from representative serial sections. Insets show magnified sections of the pericapsular region (hatched boxes) from the ventral side of the sponge (S). Muscle from the panniculus carnosus is visible in the NTD (1-35)-EGFP sponge (reddish tissue). Arrowheads in insets mark α-SMA–stained cells. Asterisk denotes a blood vessel in the muscle layer with positive α-SMA staining of the vessel wall. S indicates sponge material; GAM, gene-activated matrix; brackets in insets denote the capsular layer. Scale bar = 100 μm.

Figure 7

Collagen secretion and deposition by fibroblasts in vitro are increased by the CRT-binding sequence of TSP1. A: Sircol assay of human foreskin fibroblasts treated once daily for two days in media with 2 μmol/L ascorbic acid with 10 pmol/L TGF-β1, 10 μmol/L hep I peptide, 10 μmol/L modified hep I peptide, or media with 2 μmol/L ascorbic acid (no treatment). Results are expressed as the mean fold increase normalized to cells receiving no treatment ± SD; n = 3. Dashed lines in A–C indicate normalized value for no treatment. B: Sircol assay of human foreskin fibroblasts treated daily for two days with media with 2 μmol/L ascorbic acid and 10 nmol/L TSP1 or TSP1 incubated with either 20-fold molar excess of peptide CRT 19.36, or control peptide CRT 20.30A. Results are expressed as the mean fold increase normalized to cells receiving no treatment ± SD; n = 4. C: MEFs were treated daily for three days in media with 0.5% FBS, 2 μmol/L ascorbic acid with 30 nmol/L NoC1 with either 25-fold molar excess of peptide CRT 19.36, or control peptide CRT 20.30A. Soluble collagens were quantified by the Sircol assay. Results are expressed as the mean fold increase in soluble collagen normalized to cells receiving no treatment ± SD as analyzed by one-way analysis of variance; n = 4 separate experiments. D: MEFs were treated daily for four days in media with 2 μmol/L ascorbic acid with 10, 30, or 90 nmol/L NoC1 or with 20 μmol/L ascorbic acid. Cell layers were harvested with Laemmli buffer, separated by SDS-PAGE, and then immunoblotted with antibody to collagen III (top). Membranes were stripped and re-probed with anti-collagen I antibody (middle). Blots were stripped and reprobed with anti–β-tubulin as a loading control (bottom). *P < 0.05.

Figure 8

TSP1 binding to CRT increases collagen deposition into the insoluble extracellular matrix fraction of fibroblasts. A: MEFs were treated daily for three days in media with 0.5% FBS, 2 μmol/L ascorbic acid, and with 10 μmol/L hep I, 10 μmol/L modified hep I, 10 μmol/L scrambled hep I, 67 nmol/L TSP1, or 30 nmol/L NoC1. Cellular and pericellular protein was harvested with 1% DOC extraction (top), and the DOC-insoluble ECM was harvested with 2× SDS Laemmli buffer (bottom), separated by SDS-PAGE, and transferred to nitrocellulose membranes for immunoblotting with rabbit anti-mouse collagen I. Pro indicates procollagen; pC, procollagen with N-terminal propeptide removed, α1 (I), collagen I with both C- and N-terminal propeptides removed. B: MEFs were treated daily for three days as described above and with 30 nmol/L NoC1 in the presence or absence of 25-fold molar excess of CRT19.36 or control CRT20.30A peptide (750 nmol/L). The 1% DOC insoluble, SDS-soluble ECM was harvested, separated by SDS-PAGE, and immunoblotted for type I collagen. C: Type I collagen fibril formation in human foreskin fibroblasts was detected by immunofluorescence with rabbit anti-collagen I antibody and a Texas Red-labeled secondary antibody (red). Cell nuclei were stained with Hoechst 33342 (blue). Cells were treated daily for three days in the presence of 20 μmol/L ascorbic acid with no additional treatment or in the presence of 2 μmol/L ascorbic acid with either 5 μmol/L hep I peptide, 5 μmol/L modified hep I peptide, or media only. Scale bar = 60 μm. D: Human foreskin fibroblasts were treated daily for three days in FGM containing 0.5% FBS, 20 μmol/L ascorbic acid, and 5 μmol/L hep I peptide, 5 μmol/L modified hep I peptide, or 10 pmol/L TGF-β1. Cell matrices were separated by extraction in 4% DOC, and DOC-insoluble ECM was separated by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with rabbit anti-human collagen I. Bands were labeled as described above.

Figure 9

NoC1 does not increase active TGF-β or Smad2 phosphorylation. A: Levels of active TGF-β in the conditioned media of MEFs treated with 10 nmol/L of NoC1 or 10 nmol/L TSP1 in the presence of 2 μmol/L ascorbic acid for 48 hours were measured by PAI-1 luciferase assay (black bars). Collagen I ECM levels were calculated by densitometry of immunoblots for collagen I in the 4% DOC insoluble fraction of treated cells (gray bars). Results are reported as the fold change normalized to the mean ± SD. A representative collagen blot from this experiment is shown. B: Immunoblot for phosphorylated Smad 2 and total Smad 2/3 in MEF cell lysates from cells treated with media containing 2 μmol/L ascorbic acid and PBS, 10 nmol/L NoC1, or 8 pmol/L active TGF-β1 for 0, 1, 4, 8, and 24 hours. Cell lysates were harvested, separated by SDS-PAGE, and then immunoblotted for phosphorylated Smad 2. Blots were stripped and reprobed for total Smad 2/3.

Figure 10

An Akt inhibitor blocks collagen I protein expression in response to NoC1. MEFs cultured in media with 2 μmol/L ascorbic acid were treated daily for three days with either PI3-kinase inhibitor, 5 μmol/L LY294002 (LY), 2 μmol/L Akt inhibitor II (II), or DMSO vehicle (NT) in the presence or absence of 30 nmol/L NoC1. Cell layers harvested in Laemmli buffer were separated by SDS-PAGE and immunoblotted for type I collagen. Note that the processed and unprocessed procollagen bands were not resolved in the Laemmli extracts of the total cell lysates. Blots were stripped and reprobed for β-tubulin as a loading control. Blots were analyzed by densitometric analysis, and collagen levels were normalized to the loading control for each sample. Results are expressed as the mean fold change as compared to cells treated with DMSO ± SD (n = 3).