Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues

Abstract

Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.

Fig. 1

Western verification of periostin knockout mice. Protein lysates were generated from skin biopsies from wild-type (+/+), heterozygous (+/−), and periostin null mice (−/−), and subjected to Western analysis for periostin expression. Expression of periostin isoforms is seen around the predicted 90–100 kDa and the 37 kDa molecular weights. The heterozygotes decrease in total periostin expression by roughly half and no expression is detected in the periostin nulls. β-tubulin was used as a normalization control.

Fig. 2

Collagen and Periostin expression in adult mouse heart valve and skin. A: The entire left AV valve leaflet was micro-dissected from an adult mouse heart and stained for collagen using picrosirius red. Collagen expression is seen throughout the valve leaflet (VL), the chordae tendineae (CT) and annulus fibrosae (AF). The intense red staining indicates mature collagen fibers whereas the yellow stain shows less highly cross-linked fibers. B: The murine left AV valve leaflet was microdissected and stained in whole mount for periostin. Notice the extensive overlap in expression between periostin and collagen. C: Periostin expression in the adult mouse heart valve. Immunohistochemistry of a frontal section through an adult heart stained for periostin expression shows intense expression (green staining) within the chordae tendineae (CT). No expression is evident in the muscular left ventricle (LV) or in the papillary muscle (PM). MF20 staining was used to stain muscle (red staining). D: Immunostaining of periostin in adult mouse skin. Periostin (green staining) expression is seen throughout the various layers of the skin and is concentrated in areas surrounding the sebaceous glands (SeG) and the sweat glands (SG); HP-hypodermis.

Fig. 3

Immunogold transmission electron microscopic localization of periostin along collagen fibrils. Transverse sections of the left anterior AV valve leaflet from an adult mouse were assayed for the presence of periostin using immunogold TEM. A: No primary antibody control immunogold TEM showing no gold particles on the collagen fibers. B: Electrondense gold particles (immunogold positive dots) were localized to collagen fibrils using the anti-mouse periostin anti-sera; (Bar=500 nm).

Fig. 4

Co-Immunoprecipitation of Periostin with Collagen I. Denatured immunoprecipitated complexes were electrophoresed and immunoblotted using either an anti-HA antibody (specific for adenovirally produced periostin) (A) or an anti-collagen I antibody (B). Arrows in A (and asterisks in B) signify a positive, specific interaction with collagen type I (experimental lane). All of the negative controls (represented by I.P. controls) are negative. Positive and negative controls for the HA Western is indicated (Western Controls). To ensure that collagen I is present in the experimental lane, a reprobing of the same blot using an anti-collagen I antibody was performed. As expected, collagen immunoreactive bands are represented (arrow heads). Molecular weights are depicted at the left side of the panels.

Fig. 5

Altered distribution of collagen fibril diameter in periostin knockout and wild-type mice. A: Collagen fibril diameter was measured using NIH image as described in Materials and Methods section. A shift to the left indicates a decrease in collagen fibril diameter. B: Transmission electron micrograph of collagen fibrils from periostin knockout (−/−) and wild-type (+/+) mouse skin. Notice the decrease in fibril diameter. C, D: Hematoxylin and Eosin (H&E) staining of skin samples from wild-type (+/+) and knock-out (−/−) mice. Arrow heads denote the boundaries of the collagenous dermal layer and signifies points at which measurements were conducted. E: Graphical representations of measurements made from H&E stained sections. Notice a significant decrease in the thickness of the dermis is evident in the periostin knockout mice.

Fig. 6

Collagen cross-linking is reduced in the periostin null mouse. A: Representative differential scanning calorimetry (DSC) profiles of tendon samples from wild-type and periostin null mice. B: Thermal denaturation temperatures of wild-type and periostin null mice. Lower denaturation temperatures for null mice indicate reduced collagen cross-linking.

Fig. 7

Skin from adult periostin null mice have reduced tensile strength. A: Tensile test experimentation machine. B: Typical stress–strain relationship for adult skin sample from wild-type (+/+) and periostin null (−/−). C: Quantitative comparison of ultimate stress between the dorsal skin of wild-type (+/+) and periostin null (−/−). D: Quantitative comparison between the incremental modulus of elasticity (at the stress levels between 0.25 and 0.30 MPa) between wild-type (+/+) and periostin null (−/−) skin.

Fig. 8

Validation of periostin virus. A: Schematic of Periostin showing the fasciclin domains (Fas1–4), signal sequence (S.S.), cysteine rich domain (Cys), heparin binding domain (green ovals), putative glycosylation site, and stop codon (red asterisk). Below the periostin schematic is a representation of the two adenoviruses (LacZ and mouse periostin) generated for experimentation (HA-hemagluttinin epitope tag fused at the carboxyl-terminus of periostin, ITR-Internal terminal repeat, CMV-cytomegalovirus promoter, red asterisk-stop codon). B: Western blot for periostin (OX) or LacZ adenoviral infected HEK293 cells under reducing (with β-mercaptoethanol: β-ME) or non-reducing conditions (without β-ME). Under non-reducing conditions, periostin migrates as both a monomer and a dimer and is capable of being secreted into the supernatant. Periostin is also seen still attached to the cells, suggesting its ability to be matricellular.

Fig. 9

HH27 AV cushion infectability To verify that HH27 AV cushions in hanging drops are amenable to infection, the GFP and periostin adenoviruses were added to the cultures. A: Immunofluorescence verified infection and expression of the GFP adenovirus. B: Western blotting (using an anti-HA antibody) was performed to further confirm the infection of adenovirally produced periostin. The arrow shows immunopositive periostin expression thereby validating the adenovirus infection and expression in H27 AV cushion hanging drop cultures. Actin was used to verify equal loading.

Fig. 10

Periostin increases the visco-elastic properties of HH27 atrioventricular mesenchymal valve tissue. A: Schematic illustration of the tensiometer (not to scale), A and B correspond, respectively, to uncompressed and compressed valve explants. B: Tensiometry measurements of rounded cushion explants showing no statistical change in surface tension when periostin is overexpressed (OX). C: Fusion assays demonstrating periostin overexpression (OX) decreases the rate (proportional to the ratio of surface tension to viscosity) at which valve explants fuse. D: Statistically significant increase in viscosity of HH27 valve explants when periostin is overexpressed (OX).