Judith Litvin

The role of periostin in tissue remodeling across health and disease

Periostin, also termed osteoblast-specific factor 2, is a matricellular protein with known functions in osteology, tissue repair, oncology, cardiovascular and respiratory systems, and in various inflammatory settings. However, most of the research to date has been conducted in divergent and circumscribed areas meaning that the overall understanding of this intriguing molecule remains fragmented. Here, we integrate the available evidence on periostin expression, its normal role in development, and whether it plays a similar function during pathologic repair, regeneration, and disease in order to bring together the different research fields in which periostin investigations are ongoing. In spite of the seemingly disparate roles of periostin in health and disease, tissue remodeling as a response to insult/injury is emerging as a common functional denominator of this matricellular molecule. Periostin is transiently upregulated during cell fate changes, either physiologic or pathologic. Combining observations from various conditions, a common pattern of events can be suggested, including periostin localization during development, insult and injury, epithelial–mesenchymal transition, extracellular matrix restructuring, and remodeling. We propose mesenchymal remodeling as an overarching role for the matricellular protein periostin, across physiology and disease. Periostin may be seen as an important structural mediator, balancing appropriate versus inappropriate tissue adaption in response to insult/injury.

Expression and function of periostin-isoforms in bone.

Periostin was originally identified in MC3T3‐E1 osteoblast‐like cells. We have identified an isoform of periostin referred to as periostin‐like‐factor (PLF). It is homologous to other proteins such as fasciclin I (fas I), MPB70, βIG‐H3, and Algal‐CAMs. All of these proteins are implicated in regulating cell adhesion. PLF and the other isoforms of periostin differ in their C‐terminal sequences. PLF and periostin differ in two specific regions, between 673 and 699 amino acids (aa) and 785–812 aa. Periostin isoforms are expressed in vivo and in vitro during the stages of osteoblast differentiation and maturation. Their mRNAs are present in pre‐osteoblast cells as detected by in situ hybridization, and the proteins are between 86 and 93 kD in size as determined by Western blot analysis. Antisense oligonucleotides and antibodies directed against the isoforms of periostin were used to block the activity of these proteins. In both cases, the levels of osteoblast‐specific‐differentiation markers were markedly reduced suggesting a role for these proteins in osteoblast differentiation.

Cyclin-Dependent Kinase 4 Expression Is Essential for Neu-Induced Breast Tumorigenesis

Previous work has shown that cyclin D1 expression is required for neu- and ras-induced, but not wnt- or c-myc-induced, breast tumorigenesis in mice. Although cyclin D1 binds and activates cyclin-dependent kinase 4 (Cdk4), thereby mediating activation of a program of E2F-dependent gene expression, it has been suggested that the oncogenic activities of cyclin D1 are independent of Cdk4. To determine whether Cdk4 expression is required for breast tumorigenesis in mice, we have generated compound mice ectopically expressing the neu or wnt oncogenes in the mammary glands of wild-type and Cdk4-/- mice. Our results show that Cdk4 expression is required for efficient neu-induced tumorigenesis but is dispensable for wnt-induced breast tumorigenesis. In contrast to results previously observed in the mammary glands of cyclin D1-/- virgin females, our results show defects in mammary gland development in Cdk4-/- virgin females, suggesting differences in compensatory mechanisms in the absence of either subunit of the cyclin D1/Cdk4 complex. These results suggest that drugs targeted to inhibit Cdk4 activities could be developed to specifically treat certain breast tumors as Cdk4 is not essential for viability.

Increased expression and serum levels of the stromal cell‐secreted protein periostin in breast cancer bone metastases

Periostin, a matricellular protein, is overexpressed in the stroma of several cancers. The aim of our study was to investigate more specifically whether periostin expression is associated with bone metastases from breast cancer and to determine its source in the affected bone. Nude mice were inoculated with human MDA‐B02 breast cancer cells. Bone metastases‐bearing mice were treated with zoledronic acid—an antiresorptive drug—or vehicle. Bone metastases were examined for tumor‐ and stroma‐derived periostin expression by quantitative polymerase chain reaction with human‐ and mouse‐specific primers and immunohistochemistry. Serum periostin and conventional bone turnover markers were also measured. MDA‐B02 cells did not express periostin both in vitro and in vivo. However, mouse‐derived periostin was markedly overexpressed (eightfold) in metastatic legs compared to noninoculated mice. Serum periostin levels were also markedly increased in metastatic mice and correlated with in situ expression levels. Immunostaining showed that periostin derived from the environing stromal cells of bone metastasis. Bone turnover blockade by zoledronic acid markedly decreased osteolytic lesions but only slightly modulated serum periostin levels. Bone metastases from breast cancer induce overexpression of periostin by surrounding stromal cells. Periostin could be a biochemical marker of the early stromal response associated to breast cancer bone metastasis formation.

Exposure‐dependent increases in IL‐1β, substance P, CTGF, and tendinosis in flexor digitorum tendons with upper extremity repetitive strain injury

Upper extremity tendinopathies are associated with performance of forceful repetitive tasks. We used our rat model of repetitive strain injury to study changes induced in forelimb flexor digitorum tendons. Rats were trained to perform a high repetition high force (HRHF) handle‐pulling task (12 reaches/min at 60 ± 5% maximum pulling force [MPF]), or a low repetition negligible force (LRNF) reaching and food retrieval task (three reaches/min at 5 ± 5% MPF), for 2 h/day in 30 min sessions, 3 days/week for 3–12 weeks. Forelimb grip strength was tested. Flexor digitorum tendons were examined at midtendon at the level of the carpal tunnel for interleukin (IL)‐1β, neutrophil, and macrophage influx, Substance P, connective tissue growth factor (CTGF), and periostin‐like factor (PLF) immunoexpression, and histopathological changes. In HRHF rats, grip strength progressively decreased, while IL‐1β levels progressively increased in the flexor digitorum peritendon (para‐ and epitendon combined) and endotendon with task performance. Macrophage invasion was evident in week 6 and 12 HRHF peritendon but not endotendon. Also in HRHF rats, Substance P immunoexpression increased in week 12 peritendon as did CTGF‐ and PLF‐immunopositive fibroblasts, the increased fibroblasts contributing greatly to peritendon thickening. Endotendon collagen disorganization was evident in week 12 HRHF tendons. LRNF tendons did not differ from controls, even at 12 weeks. Thus, we observed exposure‐dependent changes in flexor digitorum tendons within the carpal tunnel, including increased inflammation, nociceptor‐related neuropeptide immunoexpression, and fibrotic histopathology, changes associated with grip strength decline.

Commitment and differentiation of cardiac myocytes

This article reviews what is known about the earliest stages of heart development focusing on the periods of commitment and differentiation of cardiac progenitor cells and their molecular regulation. The pathway from precursor to differentiated cardiac myocyte is crucial to forming a normal, functional heart. Congenital cardiac abnormalities are some of the most common, estimated at 5-8 per 1000 live births worldwide. These conditions affect mortality and morbidity of patients as infants, children, and adults. Knowledge of what steps are critical to normal heart development would lead to earlier diagnosis and possibly repair of these defects.

Periostin-like-factor in osteogenesis.

Periostin‐like‐factor (PLF), an isoform related to Periostin, is expressed in bone, heart, and vascular smooth muscle cells. PLF was detected by immunostaining in mesenchymal cells in the periosteum and in osteoblasts lining trabecular bone, suggesting that PLF has a role in osteogenesis. PLF has a signal peptide and is also secreted from osteoblasts in vitro. To study the function of PLF in osteogenesis, we assessed the effect of PLF on osteoblast proliferation and differentiation in vitro and bone formation in vivo. First, to examine whether PLF regulates osteoblast proliferation in vitro, the CyQUANT cell proliferation assay was performed. PLF over‐expression by adenovirus resulted in a significantly higher rate of cell proliferation compared to controls. This finding suggests that PLF promotes osteoblast proliferation in vitro. Second, to test whether PLF mediates osteoblast differentiation in vitro, differentiation markers of osteoblasts, were assessed, including alkaline phosphatase staining and activity, von Kossa staining and calcium deposition. Over‐expression of PLF resulted in higher expression and activity of alkaline phosphatase and higher amounts of mineralization and calcium deposition compared to controls. These data suggest that PLF promotes osteoblast differentiation in vitro. Third, to investigate the role of PLF in bone formation in vivo, PLF adenovirus was injected into 6‐week‐old rat femur bone marrow. Over‐expression of PLF resulted in increased bone formation within the marrow cavity. Lastly, in a model of fracture healing, PLF expression is robustly upregulated in callus osteoblasts at post‐fracture days 7 and 14. Taken together, these findings suggests that PLF induces bone formation in vivo. We conclude that PLF stimulates bone formation in vivo possibly by promoting osteoblast proliferation and differentiation. J. Cell. Physiol. 218: 584–592, 2009.

Immunolocalization of Periostin-like Factor and Periostin During Embryogenesis

Periostin-like factor (PLF) and Periostin are alternatively spliced mRNAs. Our findings are the first to show similarities and differences between PLF and Periostin location using isoform-specific antibodies. The differences in when and where they are present during mouse embryogenesis suggest that they may have different functions. Using immunostaining techniques, we observed that PLF was highly expressed at 12.5 days postconception (dpc) in the intermediate and outer zones of most brain regions, spinal cord, cranial and spinal nerves, and chondrocytes in developing bone and in the heart wall. By 16.5 dpc, PLF was also present in ameloblasts and odontoblasts in developing teeth, and by 19.5 dpc, PLF was present at low levels only in vagal nerve bundles, discrete white matter bundles in the brain, and chondrocytes of developing ribs. Periostin, on the other hand, was absent at 12.5 dpc from dorsal spinal cord and from cranial and spinal nerves. By 16.5 dpc, Periostin was present in many spinal nerves, but absent thereafter, and at 19.5 dpc, Periostin was present in chondrocytes in developing bone but not in neural tissues. The different spatial and temporal location of PLF and Periostin in cartilage and bone cells suggests different roles for these proteins in endochondral bone formation. The early expression of PLF in brain differentiation zones and in developing axon bundles and nerves suggests that it may facilitate axon growth.

Performance of Repetitive Tasks Induces Decreased Grip Strength and Increased Fibrogenic Proteins in Skeletal Muscle: Role of Force and Inflammation

Background This study elucidates exposure-response relationships between performance of repetitive tasks, grip strength declines, and fibrogenic-related protein changes in muscles, and their link to inflammation. Specifically, we examined forearm flexor digitorum muscles for changes in connective tissue growth factor (CTGF; a matrix protein associated with fibrosis), collagen type I (Col1; a matrix component), and transforming growth factor beta 1 (TGFB1; an upstream modulator of CTGF and collagen), in rats performing one of two repetitive tasks, with or without anti-inflammatory drugs. Methodology/Results To examine the roles of force versus repetition, rats performed either a high repetition negligible force food retrieval task (HRNF), or a high repetition high force handle-pulling task (HRHF), for up to 9 weeks, with results compared to trained only (TR-NF or TR-HF) and normal control rats. Grip strength declined with both tasks, with the greatest declines in 9-week HRHF rats. Quantitative PCR (qPCR) analyses of HRNF muscles showed increased expression of Col1 in weeks 3–9, and CTGF in weeks 6 and 9. Immunohistochemistry confirmed PCR results, and also showed greater increases of CTGF and collagen matrix in 9-week HRHF rats than 9-week HRNF rats. ELISA, and immunohistochemistry revealed greater increases of TGFB1 in TR-HF and 6-week HRHF, compared to 6-week HRNF rats. To examine the role of inflammation, results from 6-week HRHF rats were compared to rats receiving ibuprofen or anti-TNF-α treatment in HRHF weeks 4–6. Both treatments attenuated HRHF-induced increases in CTGF and fibrosis by 6 weeks of task performance. Ibuprofen attenuated TGFB1 increases and grip strength declines, matching our prior results with anti-TNFα. Conclusions/Significance Performance of highly repetitive tasks was associated with force-dependent declines in grip strength and increased fibrogenic-related proteins in flexor digitorum muscles. These changes were attenuated, at least short-term, by anti-inflammatory treatments.

The rostro-caudal position of cardiac myocytes affect their fate †

During chick embryogenesis, cells destined to form cardiac myocytes are located within the primitive streak at stage 3 in the same relative anterior‐posterior distribution as in the prelooped heart. The most rostral cells contribute to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and, retaining their relative positions, migrate to the anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the rostro‐caudal position of these cells on their diversification, anterior lateral plate mesoderm and the underlying endoderm were cut and rotated 180° along the longitudinal axis, at stages 4–8. The subsequent diversification of these precursor cells into atrial and ventricular myocytes was examined using lineage‐specific markers. Our results showed that altering location along the longitudinal axis through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate. Dev Dyn;218:123–135.