Association of TIMP-2 with extracellular matrix exposed to mechanical stress and its co-distribution with periostin during mouse mandible development

Matrix remodeling is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Periostin, originally identified in a mouse osteoblastic library, plays a role in cell adhesion and migration and in mechanical stress-induced matrix remodeling. In this study, we analyzed and compared the distribution patterns of TIMP-2 and periostin during mouse mandible development. Immunohistochemical staining for TIMP-2 and periostin was carried out on serial cryosections obtained from mice at embryonic days 13–16, postnatal day 2 (P2), P35, and 12 weeks of age. TIMP-2 and periostin exhibited a strikingly similar protein distribution during mandible development. From bud to early bell stages of molars, TIMP-2 and periostin were highly expressed on the lingual and anterior sides of the basement membrane and on the adjacent jaw mesenchyme. In pre- and postnatal incisors, the basement membrane of the apical loop and dental follicle was immunostained for TIMP-2 and periostin. At postnatal stages, TIMP-2 and periostin were prominently confined to the extracellular matrix (ECM) of gingival tissues, periodontal ligaments, and tendons (all recipients of mechanical strain). However, periostin was solely detected in the lower portion of the inner root sheath of hair follicles. Gingiva of P2 cultured in anti-TIMP-2 antibody-conditioned medium showed markedly reduced staining of periostin. We suggest that TIMP-2 and periostin are co-distributed on ECM exposed to mechanical forces and coordinately function as ECM modulators. This work was supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology and by Niigata University Research Projects.     

Cartilage proteoglycan aggregate and fibronectin can modulate the expression of type X collagen by embryonic chick chondrocytes cultured in collagen gels

Chick embryo sternal chondrocytes from the caudal and cephalic regions were cultured within type I collagen gels and type I collagen/proteoglycan aggregate composite gels in normal serum. Caudal region chondrocytes were also cultured within type I collagen gels in the presence of fibronectindepleted serum. There was a marked stimulation of type X collagen synthesis by the caudal region chondrocytes after 9 days in the presence of fibronectin-depleted serum and after 14 days in the presence of proteoglycan aggregate. These results provide evidence for the ability of chondrocytes from a zone of permanent cartilage to synthesise type X collagen and for the involvement of extracellular matrix components in the control of type X collagen gene expression.     

Estrogen-dependent expression of the tissue kallikrein gene (Klk1) in the mouse uterus and its implications for endometrial tissue growth

Tissue kallikrein mK1 is a serine protease involved in the generation of bioactive kinins for normal cardiac and arterial function in the mouse. In the present study, the tissue kallikrein gene Klk1, which codes for mK1, was shown to be one of the most prevalent of the Klk gene species in the uteri of adult mice, and its mRNA level was significantly higher at estrus than at diestrus. Klk1 mRNA expression was enhanced in the uteri of ovariectomized mice receiving estradiol-17beta treatment. Both endometrial epithelial and stromal cells isolated from the mice exhibited Klk1 expression at detectable levels when cultured in the presence of estradiol-17beta. mK1 was characterized using the recombinant active enzyme. mK1 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. Casein, gelatin, fibronectin, collagen type IV, and high-molecular-weight kininogen were degraded by mK1. The single-chain tissue-type plasminogen activator was converted to the two-chain form by mK1. In addition, mK1 degraded insulin-like growth factor binding protein-3. The present data suggest that mK1 may be implicated in the growth of uterine endometrial tissues during the proliferative phase.     

Epithelial-to-mesenchymal transition in fibrosis: Collagen type I expression is highly upregulated after EMT,but does not contribute to collagen deposition

The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-β1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-β1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.     

Mesenchymal stromal cells use PGE2 to modulate activation and proliferation of lymphocyte subsets: Combined comparison of adipose tissue, Wharton’s Jelly and bone marrow sources

Due to their immunomodulatory properties, adipose tissue (AT) and Wharton’s Jelly (WJ) constitute valuable alternatives to BM as sources of MSCs for managing graft-versus-host disease. To ensure the efficiency of AT- and WJ-MSCs implies the characterization of their immunomodulatory functions in comparison to those of BM. In this study, we investigated the capacity of AT- and WJ-MSCs to modulate lymphocyte reactions in response to different stimuli as well as the specificity of this immunomodulation. AT- and WJ-MSC displayed potent immunosuppressive effects on lymphocyte responses in a dose-dependent manner. These effects included the prevention of lymphocyte activation as well as the suppression of T-cell proliferation regardless of the stimuli used to activate lymphocytes. These effects were mediated through the expression of COX1/COX2 enzymes and by the production of PGE2. CD4⁺ and CD8⁺ T-lymphocytes were equally targeted by MSCs demonstrating that the immunomodulation was not restricted to a specific T-cell subpopulation.     

Type I collagen synthesis by human osteoblasts in response to placental lactogen and chaperonin 10, a homolog of early-pregnancy factor

Recent studies have indicated that maternal skeletal metabolism undergoes significant changes during gestation. The agents that are responsible for eliciting these changes in bone turnover during pregnancy have yet to be defined. We therefore sought to investigate whether chaperonin 10 (Cpn10), a homolog of early-pregnancy factor, or human placental lactogen (PL) were capable of influencing the synthesis of type I collagen by human osteoblasts in vitro. Both Cpn10 and PL are major components of the maternal circulation during pregnancy, but how they might contribute to bone metabolism has not been determined. Type I collagen represents the most abundant component of bone tissue, accounting for approximately 90% of the organic compartment. Both Cpn10 and PL were capable of stimulating the synthesis of type I collagen by human osteoblasts in culture. The inclusion of 17 beta-estradiol or prolactin, however, failed to influence the ability of cells to mobilize type I collagen. These novel findings support a role for PL and Cpn10 in the metabolism of bone tissue during pregnancy. Maternal bone collagen metabolism is clearly an important event during pregnancy, and the identification of the factors responsible will aid our understanding of the regulation of skeletal metabolism during gestation.     

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt,p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.     

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7⁺ satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration.