Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts.

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

Synchronized mechanical oscillations at the cell-matrix interface in the formation of tensile tissue.

The formation of uniaxial fibrous tissues with defined viscoelastic properties implies the existence of an orchestrated mechanical interaction between the cytoskeleton and the extracellular matrix. This study addresses the nature of this interaction. The hypothesis is that this mechanical interplay underpins the mechanical development of the tissue. In embryonic tendon tissue, an early event in the development of a mechanically robust tissue is the interaction of the pointed tips of extracellular collagen fibrils with the fibroblast plasma membrane to form stable interface structures (fibripositors). Here, we used a fibroblast-generated tissue that is structurally and mechanically matched to embryonic tendon to demonstrate homeostasis of cell-derived and external strain-derived tension over repeated cycles of strain and relaxation. A cell-derived oscillatory tension component is evident in this matrix construct. This oscillatory tension involves synchronization of individual cell forces across the construct and is induced in each strain cycle by transient relaxation and transient tensioning of the tissue. The cell-derived tension along with the oscillatory component is absent in the presence of blebbistatin, which disrupts actinomyosin force generation of the cell. The time period of this oscillation (60-90 s) is well-defined in each tissue sample and matches a primary viscoelastic relaxation time. We hypothesize that this mechanical oscillation of fibroblasts with plasma membrane anchored collagen fibrils is a key factor in mechanical sensing and feedback regulation in the formation of tensile tissues.

Matrix metalloproteinase 14 is required for fibrous tissue expansion.

Type I collagen-containing fibrils are major structural components of the extracellular matrix of vertebrate tissues, especially tendon, but how they are formed is not fully understood. MMP14 is a potent pericellular collagenase that can cleave type I collagen in vitro. In this study, we show that tendon development is arrested in Scleraxis-Cre::Mmp14 lox/lox mice that are unable to release collagen fibrils from plasma membrane fibripositors. In contrast to its role in collagen turnover in adult tissue, MMP14 promotes embryonic tissue formation by releasing collagen fibrils from the cell surface. Notably, the tendons grow to normal size and collagen fibril release from fibripositors occurs in Col-r/r mice that have a mutated collagen-I that is uncleavable by MMPs. Furthermore, fibronectin (not collagen-I) accumulates in the tendons of Mmp14-null mice. We propose a model for cell-regulated collagen fibril assembly during tendon development in which MMP14 cleaves a molecular bridge tethering collagen fibrils to the plasma membrane of fibripositors.

A structure-based extracellular matrix expansion mechanism of fibrous tissue growth.

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis.

Arhgap28 is a RhoGAP that inactivates RhoA and downregulates stress fibers.

The small GTPase RhoA is a major regulator of actin reorganization during the formation of stress fibers; thus identifying molecules that regulate Rho activity is necessary for a complete understanding of the mechanisms that determine cell contractility. Here, we have identified Arhgap28 as a Rho GTPase activating protein (RhoGAP) that switches RhoA to its inactive form. We generated an Arhgap28-LacZ reporter mouse that revealed gene expression in soft tissues at E12.5, pre-bone structures of the limb at E15.5, and prominent expression restricted mostly to ribs and limb long bones at E18.5 days of development. Expression of recombinant Arhgap28-V5 in human osteosarcoma SaOS-2 cells caused a reduction in the basal level of RhoA activation and disruption of actin stress fibers. Extracellular matrix assembly studies using a 3-dimensional cell culture system showed that Arhgap28 was upregulated during Rho-dependent assembly of the ECM. Taken together, these observations led to the hypothesis that an Arhgap28 knockout mouse model would show a connective tissue phenotype, perhaps affecting bone. Arhgap28-null mice were viable and appeared normal, suggesting that there could be compensation from other RhoGAPs. Indeed, we showed that expression of Arhgap6 (a closely related RhoGAP) was upregulated in Arhgap28-null bone tissue. An upregulation in RhoA expression was also detected suggesting that Arhgap28 may be able to additionally regulate Rho signaling at a transcriptional level. Microarray analyses revealed that Col2a1, Col9a1, Matn3, and Comp that encode extracellular matrix proteins were downregulated in Arhgap28-null bone. Although mutations in these genes cause bone dysplasias no bone phenotype was detected in the Arhgap-28 null mice. Together, these data suggest that the regulation of Rho by RhoGAPs, including Arhgap28, during the assembly and development of mechanically strong tissues is complex and may involve multiple RhoGAPs.

Tendon is covered by a basement membrane epithelium that is required for cell retention and the prevention of adhesion formation.

The ability of tendons to glide smoothly during muscle contraction is impaired after injury by fibrous adhesions that form between the damaged tendon surface and surrounding tissues. To understand how adhesions form we incubated excised tendons in fibrin gels (to mimic the homeostatic environment at the injury site) and assessed cell migration. We noticed cells exiting the tendon from only the cut ends. Furthermore, treatment of the tendon with trypsin resulted in cell extravagation from the shaft of the tendons. Electron microscopy and immunolocalisation studies showed that the tendons are covered by a novel cell layer in which a collagen type IV/laminin basement membrane (BM) overlies a keratinised epithelium. PCR and western blot analyses confirmed the expression of laminin ß1 in surface cells, only. To evaluate the cell retentive properties of the BM in vivo we examined the tendons of the Col4a1(+/Svc) mouse that is heterozygous for a G-to-A transition in the Col4a1 gene that produces a G1064D substitution in the α1(IV) chain of collagen IV. The flexor tendons had a discontinuous BM, developed fibrous adhesions with overlying tissues, and were acellular at sites of adhesion formation. In further experiments, tenotomy of wild-type mice resulted in expression of laminin throughout the adhesion. In conclusion, we show the existence of a novel tendon BM-epithelium that is required to prevent adhesion formation. The Col4a1(+/Svc) mouse is an effective animal model for studying adhesion formation because of the presence of a structurally-defective collagen type IV-containing BM.