The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.

Role of the CCN protein family in cancer.

The CCN protein family is composed of six matricellular proteins, which serve regulatory roles rather than structural roles in the extracellular matrix. First identified as secreted proteins which are induced by oncogenes, the acronym CCN came from the names of the first three members: CYR61, CTGF, and NOV. All six members of the CCN family consist of four cysteine-rich modular domains. CCN proteins are known to regulate cell adhesion, proliferation, differentiation, and apoptosis. In addition, CCN proteins are associated with cardiovascular and skeletal development, injury repair, inflammation, and cancer. They function either through binding to integrin receptors or by regulating the expression and activity of growth factors and cytokines. Given their diverse roles related to the pathology of certain diseases such as fibrosis, arthritis, atherosclerosis, diabetic nephropathy, retinopathy, and cancer, there are many emerging studies targeting CCN protein signaling pathways in attempts to elucidate their potentials as therapeutic targets. [BMB Reports 2018; 51(10): 486-493].

The CCN Proteins: An Overview.

I introduce the general structures and functions of CCN proteins and possible molecular mechanisms regarding the unique biological actions of this new family of signaling regulators, which may be referred to as "signal conductors." Relevance to pathology is also briefly introduced. The information provided in this overview should be useful for readers of the following chapters.

Western Blotting Analysis of CCN Proteins in Calcified Tissues.

Western blotting is widely used for protein analysis. We routinely perform such analysis for evaluating the production levels of CCN family proteins in a variety of cells under various conditions. In this chapter, we describe our Western blotting protocol to estimate protein production profiles of CCN family members after having assessed the specificity of the antibodies against each CCN member protein to ensure no cross-reaction with other CCN member proteins.

Preparation of Module-Specific Antibodies Against CCN Family Members.

Specific antibodies against biomolecules are conventional, but robust tools for the structural and functional analysis of target molecules. Since CCN family proteins are composed of four distinct modules that together determine the functionalities as full-length molecules depending upon extracellular microenvironment, specific antibody against independent modules are quite useful in CCN family research. Three distinct strategies are considerable for raising antibodies specific to four modules: IGFBP, VWC, TSP1, and CT modules. In the first strategy, full-length CCN family proteins are used to immunize mice to obtain a number of hybridoma clones producing different monoclonal antibodies, which are to be characterized to locate the epitopes in particular modules. Second methodology is a straightforward one, in which each modular protein fragment or synthetic peptide is prepared and is used for the immunization of animals independently. Finally, DNA immunization technology is recently known to be useful in developing module-specific antibodies against CCN family proteins as well. Preparation of antibodies is a quite classical and established technique, and thus nowadays is managed mostly by professional and commercial facilities. Therefore in this chapter, essentials of each strategy are introduced, rather than experimental details in each process.

Protocols for Screening Peptide Motifs Binding to CCN Family Proteins.

Function of CCN family proteins is determined by the interactions with multiple cofactors that are present in microenvironment. Therefore, finding out these cofactors is critically important in understanding the molecular function of the CCN family members. For this objective, bacteriophage random peptide display library is a quite feasible tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of random 12-16 amino acid residues on its surface. Bacteriophage clones that possess the peptides that bind to a CCN family protein are selected through several cycles of a process designated biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, oligopeptides that specifically bind to the CCN family member can be specified. Obtained peptide sequences can be utilized for designing peptide aptamers for the CCN family protein, or as a key sequence to find out new CCN family cofactor candidates in silico.

Degradomic and yeast 2-hybrid inactive catalytic domain substrate trapping identifies new membrane-type 1 matrix metalloproteinase (MMP14) substrates: CCN3 (Nov) and CCN5 (WISP2).

Members of the CCN family of matricellular proteins are cytokines linking cells to the extracellular matrix. We report that CCN3 (Nov) and CCN5 (WISP2) are novel substrates of MMP14 (membrane-type 1-matrix metalloproteinase, MT1-MMP) that we identified using MMP14 "inactive catalytic domain capture" (ICDC) as a yeast two-hybrid protease substrate trapping platform in parallel with degradomics mass spectrometry screens for MMP14 substrates. CCN3 and CCN5, previously unknown substrates of MMPs, were biochemically validated as substrates of MMP14 and other MMPs in vitro-CCN5 was processed in the variable region by MMP14 and MMP2, as well as by MMP1, 3, 7, 8, 9 and 15. CCN1, 2 and 3 are proangiogenic factors yet we found novel opposing activity of CCN5 that was potently antiangiogenic in an aortic ring vessel outgrowth model. MMP14, a known regulator of angiogenesis, cleaved CCN5 and abrogated the angiostatic activity. CCN3 was also processed in the variable region by MMP14 and MMP2, and by MMP1, 8 and 9. In addition to the previously reported cleavages of CCN1 and CCN2 by several MMPs we found that MMPs 8, 9, and 1 process CCN1, and MMP8 and MMP9 also process CCN2. Thus, our study reveals additional and pervasive family-wide processing of CCN matricellular proteins/cytokines by MMPs. Furthermore, CCN5 cleavage by proangiogenic MMPs results in removal of an angiogenic brake held by CCN5. This highlights the importance of thorough dissection of MMP substrates that is needed to reveal higher-level control mechanisms beyond type IV collagen and other extracellular matrix protein remodelling in angiogenesis. SUMMARY: We find CCN family member cleavage by MMPs is more pervasive than previously reported and includes CCN3 (Nov) and CCN5 (WISP2). CCN5 is a novel antiangiogenic factor, whose function is abrogated by proangiogenic MMP cleavage. By processing CCN proteins, MMPs regulate cell responses angiogenesis in connective tissues.

CCN4/WISP1 (WNT1 inducible signaling pathway protein 1): a focus on its role in cancer.

The matricellular protein WISP1 is a member of the CCN protein family. It is induced by WNT1 and is a downstream target of ß-catenin. WISP1 is expressed during embryonic development, wound healing and tissue repair. Aberrant WISP1 expression is associated with various pathologies including osteoarthritis, fibrosis and cancer. Its role in tumor progression and clinical outcome makes WISP1 an emerging candidate for the detection and treatment of tumors.

The CCN family acting throughout the body: recent research developments.

The animal body is composed of a variety of cells and extracellular matrices that are organized and orchestrated in a harmonized manner to support life. Therefore, the critical importance of a comprehensive understanding of the molecular network surrounding and integrating the cells is now emphasized. The CCN family is a novel group of matricellular proteins that interact with and orchestrate a number of extracellular signaling and matrix molecules to construct and maintain living tissues. This family comprises six distinct members in mammals, which are characterized by a unique and conserved modular structure. These proteins are not targeted to limited and specific receptors to execute specific missions, but manipulate a vast number of biomolecules in the network by serving as a molecular hub at the center. The unified nomenclature, CCN, originates from a simple acronym of the three classical members, which helps us to avoid having any preconception about their pleiotropic and anonymous functional nature. In this review, after a brief summary of the general molecular concepts regarding the CCN family, new aspects of each member uncovered by recent research are introduced, which represent, nevertheless, only the tip of the iceberg of the profound functionality of these molecules.

Analysis of the WISP3 gene in Indian families with progressive pseudorheumatoid dysplasia.

Progressive pseudorheumatoid dysplasia (PPD) is a progressive skeletal syndrome characterized by stiffness, swelling and pain in multiple joints with associated osteoporosis in affected patients. Radiographically, the predominant features resemble a spondyloepiphyseal dysplasia. Mutations in the WISP3 gene are known to cause this autosomal recessive condition. To date, only a limited number of studies have looked into the spectrum of mutations causing PPD. We report on clinical features and WISP3 mutations in a large series of Indian patients with this rare skeletal dysplasia. Families with at least one member showing clinical and radiologic features of PPD were recruited for the study. Symptoms, signs and radiographic findings were documented in 35 patients from 25 unrelated families. Swelling of small joints of hands and contractures are the most common presenting features. Mutation analysis was carried out by bidirectional sequencing of the WISP3 gene in all 35 patients. We summarize the clinical features of 35 patients with PPD and report on 11 different homozygous mutations and one instance of compound heterozygosity. Eight (c.233G>A, c.340T>C, c.348C>A, c.433T>C, c.682T>C, c.802T>G, c.947_951delAATTT, and c.1010G>A) are novel mutations and three (c.156C>A, c.248G>A, and c.739_740delTG) have been reported previously. One missense mutation (c.1010G>A; p.Cys337Tyr) appears to be the most common in our population being seen in 10 unrelated families. This is the largest cohort of patients with PPD in the literature and the first report from India on mutation analysis of WISP3. We also review all the mutations reported in WISP3 till date.

Spiked-in pulsed in vivo labeling identifies a new member of the CCN family in regenerating newt hearts.

The newt Notophthalmus viridescens , which belongs to the family of salamanders (Urodela), owns remarkable regenerative capacities allowing efficient scar-free repair of various organs including the heart. Salamanders can regrow large parts of the myocardium unlike mammals, which cannot replace lost cardiomyocytes efficiently. Unfortunately, very little is known about the molecules and the regulatory circuits facilitating efficient heart regeneration in newts or salamanders. To identify proteins that are involved in heart regeneration, we have developed a pulsed SILAC-based mass spectrometry method based on the detection of paired peptide peaks after ¹³C6-lysine incorporation into proteins in vivo. Proteins were identified by matching mass spectrometry derived peptide sequences to a recently established normalized newt EST library. Our approach enabled us to identify more than 2200 nonredundant proteins in the regenerating newt heart. Because of the pulsed in vivo labeling approach, accurate quantification was achieved for 1353 proteins, of which 72 were up- and 31 down-regulated with a (|log 2 ratio| > 1) during heart regeneration. One deregulated member was identified as a new member of the CCN protein family, showing a wound specific activation. We reason that the detection of such deregulated newt-specific proteins in regenerating hearts supports the idea of a local evolution of tissue regeneration in salamanders. Our results significantly improve understanding of dynamic changes in the complex protein network that underlies heart regeneration and provides a basis for further mechanistic studies.

The diagnostic challenge of progressive pseudorheumatoid dysplasia (PPRD): a review of clinical features, radiographic features, and WISP3 mutations in 63 affected individuals.

Progressive pseudorheumatoid dysplasia (PPRD) is a genetic, non-inflammatory arthropathy caused by recessive loss of function mutations in WISP3 (Wnt1-inducible signaling pathway protein 3; MIM 603400), encoding for a signaling protein. The disease is clinically silent at birth and in infancy. It manifests between the age of 3 and 6 years with joint pain and progressive joint stiffness. Affected children are referred to pediatric rheumatologists and orthopedic surgeons; however, signs of inflammation are absent and anti-inflammatory treatment is of little help. Bony enlargement at the interphalangeal joints progresses leading to camptodactyly. Spine involvement develops in late childhood and adolescence leading to short trunk with thoracolumbar kyphosis. Adult height is usually below the 3rd percentile. Radiographic signs are relatively mild. Platyspondyly develops in late childhood and can be the first clue to the diagnosis. Enlargement of the phalangeal metaphyses develops subtly and is usually recognizable by 10 years. The femoral heads are large and the acetabulum forms a distinct "lip" overriding the femoral head. There is a progressive narrowing of all articular spaces as articular cartilage is lost. Medical management of PPRD remains symptomatic and relies on pain medication. Hip joint replacement surgery in early adulthood is effective in reducing pain and maintaining mobility and can be recommended. Subsequent knee joint replacement is a further option. Mutation analysis of WISP3 allowed the confirmation of the diagnosis in 63 out of 64 typical cases in our series. Intronic mutations in WISP3 leading to splicing aberrations can be detected only in cDNA from fibroblasts and therefore a skin biopsy is indicated when genomic analysis fails to reveal mutations in individuals with otherwise typical signs and symptoms. In spite of the first symptoms appearing in early childhood, the diagnosis of PPRD is most often made only in the second decade and affected children often receive unnecessary anti-inflammatory and immunosuppressive treatments. Increasing awareness of PPRD appears to be essential to allow for a timely diagnosis.

Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal.

The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.

Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets.

Members of the CCN family of matricellular proteins are crucial for embryonic development and have important roles in inflammation, wound healing and injury repair in adulthood. Deregulation of CCN protein expression or activities contributes to the pathobiology of various diseases - many of which may arise when inflammation or tissue injury becomes chronic - including fibrosis, atherosclerosis, arthritis and cancer, as well as diabetic nephropathy and retinopathy. Emerging studies indicate that targeting CCN protein expression or signalling pathways holds promise in the development of diagnostics and therapeutics for such diseases. This Review summarizes the biology of CCN proteins, their roles in various pathologies and their potential as therapeutic targets.

Cloning, expression and purification of the CCN family of proteins in Escherichia coli.

The CCN proteins are extracellular matrix associated proteins involved in critical cell activities and several aggressive forms of cancer. The proteins share a modular structure of four discrete domains and 38 conserved cysteine residues. The absence of any structural information of these proteins has resulted in a need for the ability to produce substantial amounts of pure CCN protein. Through bacterial expression and inclusion body based purification, pure recombinant CCN proteins have been produced for use in structural and biochemical experiments.

CCN proteins in normal and injured liver.

CCN proteins are small secreted cysteine-rich proteins containing up to four individual structural modules including an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, a thrombospondin type I module and a carboxyl-terminal cystine knot. Actually, there is a large body of evidence suggesting that members of the CCN protein family encompass an expansive repertoire of functions in crucial areas including control of development, cell fate, angiogenesis, tumorigenesis, osteogenesis, cell adhesion, mitogenesis, migration, chemotaxis, and cell survival. Moreover, this family is supposed to modulate signalling of integrins, transforming growth factor-betas, bone morphogenetic proteins, vascular endothelial growth factor, Notch and factors that mediate signals via the canonical Wingless-type MMTV integration site family. However, several of these properties are not substantiated by experimental data but were deduced from proteins sharing one or more of the structural modules with these proteins. In this review, the actual knowledge of biological activities and molecular involvement of CCN proteins in maintenance of liver health and in initiation and progression of hepatic diseases is summarized and discussed.

The CCN family of proteins: structure-function relationships.

The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure-function relationships.