CTGF Human, HEK

What is CTGF and why is it significant in human cellular research?

CTGF (Connective Tissue Growth Factor), also designated CCN2, is a secreted matricellular protein belonging to the CCN family that plays crucial roles in multiple cellular processes. CTGF has been identified as a potential biomarker for fibrotic diseases due to its involvement in tissue scarring processes. As a downstream mediator of TGF-β1 signaling, CTGF stimulates interstitial deposition of extracellular matrix (ECM) proteins and promotes proliferation of various cell types . The protein's multifunctional nature makes it relevant to research in fibrosis, cancer, wound healing, and various pathological conditions. Unlike its family member CCN3/NOV which exhibits growth-inhibiting properties, CTGF generally promotes proliferative responses, highlighting the interesting counterregulatory functions within this protein family . Understanding CTGF's multifaceted biological activities provides critical insights into the molecular mechanisms of tissue remodeling and fibrotic disorders.

What is the molecular structure of human CTGF and its functional domains?

Human CTGF is a modular protein composed of four conserved domains that contribute to its diverse functions. These domains include: (1) an insulin-like growth factor binding protein domain (IGFBP), (2) a von Willebrand factor type C repeat (VWC), (3) a thrombospondin type I repeat (TSP-1), and (4) a C-terminal (CT) domain containing a cystine knot motif. The thrombospondin type I repeat has been identified as a cell attachment motif that plays a crucial role in protein-protein interactions. Research has demonstrated that this domain specifically mediates CTGF's interaction with estrogen receptors (ER) . The modular structure enables CTGF to interact with multiple signaling molecules and cell surface receptors, explaining its pleiotropic effects on cell adhesion, migration, proliferation, and ECM production. Truncation experiments have shown that the CTGF mutant lacking the ER-binding site (CTGF(1-187)) completely abolishes CTGF's capacity to repress estrogen-responsive transcription .

How are HEK293 cells utilized for CTGF expression studies?

HEK293 cells serve as an excellent expression platform for recombinant human CTGF production due to their high transfection efficiency, robust protein synthesis machinery, and capacity for proper post-translational modifications. Researchers have successfully established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF . These stable cell lines offer consistent and scalable production of CTGF, making them valuable tools for studying CTGF's biological activities. Additionally, HEK293 cells have been used to investigate CTGF's functional effects by transfection with eukaryotic expression plasmids containing coding sequences of human CTGF . In such systems, secreted CTGF can be isolated and purified using chromatography techniques for subsequent functional studies. The HEK293-MAS system has also been employed to study the relationship between CTGF and the MAS receptor, demonstrating that CTGF expression is essential for MAS-mediated up-regulation of different collagen subtype genes .

What post-translational modifications occur in CTGF when expressed in HEK cells?

Research has demonstrated that recombinant CTGF expressed in HEK cell systems undergoes N-glycosylation, a critical post-translational modification that may affect protein folding, stability, and biological function. This finding, first reported by Bohr et al. (2010), represents an important contribution to understanding CTGF biology . The presence of N-glycosylation may influence CTGF's interaction with other proteins and its extracellular signaling capabilities. In HEK expression systems, researchers have confirmed the identity of purified human CCN2/CTGF using in-gel digest followed by ESI-TOF/MS mass spectrometry, which can detect these modifications . Understanding these post-translational modifications is crucial for researchers working with recombinant CTGF, as they may impact the protein's biological activities in experimental settings. Properly glycosylated CTGF produced in mammalian systems like HEK cells likely better represents the native conformation of the protein compared to prokaryotic expression systems.

What are optimal methods for recombinant CTGF expression in HEK cells?

For optimal recombinant CTGF expression in HEK cells, researchers have employed several successful strategies. Establishing stable HEK and Flp-In-293 clones has proven to be an effective approach for consistent production of human CCN2/CTGF . The Flp-In system allows for site-specific integration of the expression construct, ensuring uniform expression levels across the cell population. For transient expression, HEK293 cells can be transfected with eukaryotic expression plasmids containing human CTGF coding sequences . The use of strong promoters (such as CMV) and inclusion of appropriate secretion signal sequences ensures efficient expression and secretion of the protein. For applications requiring higher expression levels, adenoviral vector systems have also been developed, similar to those used for recombinant expression of the related CCN3/NOV protein . When designing expression constructs, researchers should consider including affinity tags (such as His or FLAG) to facilitate subsequent purification while confirming that such modifications do not interfere with protein activity through appropriate functional assays.

How can researchers purify recombinant CTGF from HEK cell cultures?

Purification of recombinant CTGF from HEK cell cultures typically involves a multi-step chromatographic approach. Researchers have established protocols to purify large quantities of CCN proteins that maintain their biological activity . The process begins with collection of conditioned media from CTGF-expressing HEK cells, followed by clarification through centrifugation and/or filtration to remove cellular debris. For His-tagged CTGF, immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins serves as an effective initial capture step. Further purification often employs ion exchange chromatography to separate CTGF from remaining contaminants based on charge differences. Size exclusion chromatography provides a final polishing step, allowing separation of monomeric CTGF from aggregates and ensuring high purity. The identity of purified human CCN2/CTGF can be confirmed through in-gel digest followed by ESI-TOF/MS mass spectrometry . Throughout the purification process, maintaining protein stability through appropriate buffer conditions (pH, salt concentration) and addition of protease inhibitors is essential for preserving CTGF's biological activity.

How can researchers verify the biological activity of purified CTGF?

Verification of purified CTGF's biological activity is essential before conducting further experiments. Several validated functional assays have been employed in the literature. The Smad3-sensitive reporter gene assay provides a direct measure of CTGF's ability to modulate TGF-β signaling pathways, which is central to its biological function . The BrdU proliferation assay in cell lines such as EA- hy 926 cells can assess CTGF's effect on cellular proliferation . For evaluating CTGF's impact on extracellular matrix production, measuring expression levels of ECM components like fibronectin and various collagen subtypes (I, III, IV, VI) via real-time RT-PCR and western blot analyses serves as a reliable indicator . Additionally, investigating CTGF's effect on integrin expression, particularly subunits αV and β1, can provide insights into its functional activity . For studies focusing on CTGF's interaction with estrogen receptors, transcriptional repression of estrogen-responsive genes such as pS2 and cathepsin D can be assessed through reporter assays and protein expression analysis .

What challenges exist in producing functionally active CTGF in HEK systems?

Producing functionally active CTGF in HEK systems presents several challenges that researchers must navigate. A primary consideration is maintaining protein integrity, as CTGF's complex disulfide bonding pattern is crucial for its proper folding and function. Overexpression can sometimes lead to protein misfolding or aggregation, necessitating optimization of expression conditions. The N-glycosylation pattern observed in CTGF expressed in HEK cells must be preserved during purification, as alterations in these post-translational modifications may affect biological activity. Additionally, CTGF's tendency to interact with extracellular matrix components can complicate purification efforts. Researchers must also be mindful that CTGF's biological effects are concentration-dependent, with studies showing that lower concentrations can cause autoinduction of CTGF expression . This suggests that expression levels need careful calibration for functional studies. Finally, as a secreted protein, CTGF requires its signal peptide for proper processing and secretion, as demonstrated by studies showing that CTGF without its signal peptide (CTGF(Δ1-26)) cannot be secreted into medium and loses its ability to repress estrogen receptor transcriptional activity .

How does CTGF mediate extracellular matrix regulation in human cells?

CTGF plays a pivotal role in extracellular matrix regulation by modulating the expression and deposition of various ECM components. Studies with human trabecular meshwork (TM) cells have demonstrated that CTGF treatment increases the expression of fibronectin, collagen types I, III, IV, and VI, as well as the integrin subunits αV and β1 . This CTGF-induced ECM production contributes to tissue remodeling and, in pathological conditions, can lead to fibrosis. CTGF functions as a downstream mediator of TGF-β2 signaling, which is implicated in the increased ECM deposition observed in primary open-angle glaucoma (POAG) . Interestingly, CTGF exhibits concentration-dependent effects, with lower concentrations causing autoinduction of CTGF expression, creating a feed-forward loop that can amplify ECM production . Unlike its effects on matrix proteins, CTGF does not appear to significantly impact the expression and activity of matrix metalloproteinases (MMP-2, MMP-9) or plasminogen activator inhibitor-1 (PAI-1) . This selective regulation of ECM components without affecting degradation pathways suggests that CTGF primarily promotes matrix accumulation rather than inhibiting matrix turnover.

What is the relationship between CTGF and TGF-β signaling?

CTGF functions as a critical downstream mediator of TGF-β signaling, forming an important regulatory axis in fibrotic processes and tissue remodeling. TGF-β2 induces CTGF expression, which then contributes to extracellular matrix production and cellular proliferation . This relationship has been demonstrated in human trabecular meshwork cells, where TGF-β2 treatment increases CTGF expression, leading to enhanced ECM deposition characteristic of primary open-angle glaucoma . The functional significance of this relationship is evident from siRNA experiments, where CTGF-specific siRNA inhibits the TGF-β2-induced upregulation of fibronectin, confirming CTGF's role as a mediator of TGF-β2 effects on ECM synthesis . The biological activity of purified CTGF can be assessed using Smad3-sensitive reporter gene assays, reflecting its involvement in the canonical TGF-β signaling pathway . This TGF-β/CTGF axis represents a potential intervention point for therapeutic strategies aimed at preventing or reversing pathological structural changes in fibrotic diseases, as pharmacological modulation of CTGF might interfere with the downstream effects of TGF-β signaling without completely abolishing this essential pathway's other functions .

How does CTGF interact with the MAS receptor to regulate collagen gene expression?

CTGF serves as a critical mediator in the MAS receptor's regulation of collagen gene expression, revealing a complex signaling network involved in cardiac fibrosis. Research has demonstrated that expression of the CTGF gene is essential for MAS-mediated up-regulation of different collagen subtype genes in both HEK293-MAS cells and human cardiac fibroblasts . The MAS receptor, a G protein-coupled receptor, signals through the ERK1/2 pathway to increase both mRNA and protein levels of CTGF . This signaling cascade can be specifically blocked by MAS inverse agonist AR244555 and by MEK1 inhibition, confirming the specificity of this pathway . The functional significance of this interaction is highlighted by RNAi experiments, where knockdown of CTGF disrupts collagen gene regulation by MAS agonists . Interestingly, elevated expression of MAS, CTGF, and collagen genes has been observed in failing human heart samples compared to non-failing samples, and expression levels of MAS correlate with CTGF in both conditions . This MAS-CTGF-collagen pathway represents a potential target for pharmacological intervention in heart failure, suggesting that blocking this specific signaling cascade might help mitigate cardiac fibrosis.

How does CTGF interact with estrogen receptors (ER)?

CTGF physically and functionally interacts with estrogen receptors, revealing an unexpected role in modulating estrogen signaling. Research has demonstrated that CTGF can directly bind to ER both in vitro and in vivo, with the interaction occurring specifically at the ER DNA-binding domain . The ER interaction region in CTGF has been mapped to the thrombospondin type I repeat, which is a cell attachment motif . This physical interaction has functional consequences, as overexpression of CTGF inhibits ER transcriptional activity and suppresses the expression of estrogen-responsive genes, including pS2 and cathepsin D . Conversely, reduction of endogenous CTGF with CTGF small interfering RNA enhances ER transcriptional activity . The importance of this interaction is further highlighted by experiments with CTGF mutants lacking the ER-binding site, which abolish CTGF's ability to repress estrogen-responsive transcription . Additionally, the secreted form of CTGF, rather than the cytoplasmic form, is responsible for repression of ER transcriptional activity, as demonstrated by experiments with CTGF lacking its signal peptide (CTGF(Δ1-26)) . This CTGF-ER interaction provides a novel mechanism for cross-talk between secreted growth factor and estrogen receptor signaling pathways.

What techniques are available for modulating CTGF expression in HEK cells?

Researchers have several effective techniques for modulating CTGF expression in HEK cells, each with specific applications. For overexpression studies, transfection with eukaryotic expression plasmids containing human CTGF coding sequences has been successfully employed . When stable expression is desired, researchers have established HEK and Flp-In-293 clones that serve as productive sources for recombinant human CTGF . For transient high-level expression, adenoviral vector systems can be utilized, similar to those developed for the related protein CCN3/NOV . For knockdown experiments, RNA interference approaches using vector-based CTGF siRNAs have proven effective in reducing endogenous CTGF expression . These siRNA approaches have been valuable for demonstrating CTGF's role in mediating TGF-β2 effects on ECM synthesis and in regulating ER transcriptional activity . For structure-function studies, expression constructs encoding mutant forms of CTGF have been developed, including truncation mutants lacking specific domains (such as CTGF(1-187)) and mutants lacking the signal peptide (CTGF(Δ1-26)) . These tools enable precise interrogation of domain-specific functions and the importance of CTGF secretion for its biological activities.

What are the optimal methods for detecting and quantifying CTGF expression?

Accurate detection and quantification of CTGF expression is crucial for experimental reproducibility. At the mRNA level, real-time RT-PCR provides sensitive quantification of CTGF transcript levels and has been used to demonstrate CTGF autoinduction at lower concentrations and to assess the effects of CTGF silencing on TGF-β2-induced gene expression . For protein detection, Western blot analysis using specific antibodies against CTGF or epitope tags (such as FLAG) in recombinant constructs allows visualization and semi-quantitative analysis of expression levels . For secreted CTGF, analysis of conditioned media is essential, as demonstrated in studies showing that CTGF without its signal peptide fails to be secreted . Immunoprecipitation techniques have been employed to detect protein-protein interactions, such as the physical association between CTGF and ER . For functional assessment, reporter gene assays using Smad3-sensitive reporters can measure CTGF's ability to modulate TGF-β signaling , while ERE-Luc (estrogen-responsive element-containing luciferase) reporters can assess CTGF's effect on ER transcriptional activity . Mass spectrometry, particularly ESI-TOF/MS following in-gel digest, provides definitive identification of purified CTGF and can detect post-translational modifications such as N-glycosylation .

How can researchers assess CTGF-mediated effects on extracellular matrix production?

Assessment of CTGF's effects on extracellular matrix production requires a multi-parameter approach examining both gene expression and protein levels. Real-time RT-PCR analysis of ECM component genes provides a sensitive measure of CTGF's impact on matrix production, with studies showing that CTGF treatment increases expression of fibronectin, collagen types I, III, IV, and VI in human trabecular meshwork cells . Western blot analysis complements these findings by confirming changes at the protein level. For a more comprehensive evaluation, researchers can also assess the expression of integrin subunits (αV and β1), which mediate cell-matrix interactions and are upregulated by CTGF . To determine whether CTGF affects matrix degradation pathways, gelatine zymography can be used to analyze the activity of matrix metalloproteinases (MMPs), though studies suggest CTGF does not significantly impact MMP-2 and MMP-9 activity . The functional relationship between CTGF and other signaling pathways can be investigated using specific inhibitors or siRNA approaches, as demonstrated by experiments showing that CTGF-specific siRNA inhibits TGF-β2-induced upregulation of fibronectin . In cardiac fibroblast models, knockdown of CTGF by RNAi disrupts collagen gene regulation by MAS receptor agonists, confirming CTGF's essential role in this process .

What controls should be included when studying CTGF-mediated cellular responses?

When designing experiments to study CTGF-mediated cellular responses, several critical controls should be incorporated to ensure reliable and interpretable results. For recombinant CTGF studies, protein preparation controls are essential, including verification of protein purity by SDS-PAGE and confirmation of identity by mass spectrometry, as demonstrated in studies using ESI-TOF/MS . Concentration-response experiments are necessary given that CTGF exhibits dose-dependent effects, with lower concentrations causing autoinduction of CTGF expression . When investigating CTGF's role in mediating effects of upstream factors like TGF-β2 or MAS receptor signaling, specific inhibitors (such as MEK1 inhibition for the ERK1/2 pathway) and siRNA approaches provide valuable mechanistic controls . For CTGF overexpression studies, appropriate vector-only controls must be included, while domain-specific mutants (such as CTGF(1-187) lacking the ER-binding site) can serve as functional controls to demonstrate the specificity of observed effects . When studying CTGF's impact on nuclear receptor signaling, specificity controls examining effects on other nuclear receptors (such as androgen receptor and glucocorticoid receptor) help establish whether CTGF selectively regulates specific pathways . Time-course experiments are also valuable for understanding the dynamics of CTGF-mediated responses, particularly when examining downstream effects on gene expression or cellular processes.

How can CTGF research in HEK cells be translated to disease-relevant models?

Translating CTGF research from HEK cells to disease-relevant models requires systematic approaches that bridge fundamental molecular insights with physiological contexts. While HEK cells provide valuable platforms for recombinant protein production and mechanistic studies, disease-specific cell types better represent pathological processes. For example, human trabecular meshwork cells have been used to investigate CTGF's role in glaucoma-related extracellular matrix changes , while human cardiac fibroblasts serve as models for studying CTGF's involvement in heart failure and fibrosis . Co-culture systems incorporating multiple cell types can better recapitulate tissue microenvironments where CTGF functions. Regarding in vivo translation, conditional transgenic models with tissue-specific CTGF overexpression or knockout allow examination of CTGF's roles in specific pathological contexts. For therapeutic development, the identification of the MAS-CTGF-collagen pathway suggests potential targets for pharmacological intervention in heart failure , while CTGF's interaction with estrogen receptors may have implications for hormone-responsive cancers . As CTGF exhibits organ-specific and context-dependent functions, comprehensive analysis across multiple disease models is necessary to develop targeted interventions based on its role as a TGF-β mediator in fibrotic processes.

What are the current limitations in CTGF functional studies?

Despite significant advances, several limitations persist in CTGF functional studies that researchers should consider when designing experiments and interpreting results. The multifunctional nature of CTGF, with its four distinct domains mediating various protein-protein interactions, creates complexity in attributing specific cellular responses to particular structural elements. While domain-specific mutants provide valuable insights, they may not fully recapitulate the integrated functions of the intact protein. Additionally, CTGF's concentration-dependent effects, including autoinduction at lower concentrations , necessitate careful calibration of experimental conditions. The post-translational modifications of CTGF, particularly N-glycosylation , may vary between expression systems and affect functional outcomes, potentially limiting the comparability of results across studies. The distinction between secreted and intracellular CTGF functions, highlighted by experiments with signal peptide-deleted mutants , adds another layer of complexity. Furthermore, CTGF's interactions with multiple signaling pathways, including TGF-β and MAS receptor signaling , create challenges in isolating its direct effects from secondary responses mediated by these interacting pathways. Finally, the translation of findings from cell culture models to in vivo pathological processes requires consideration of tissue-specific contexts and the complex multicellular environments where CTGF functions.

How might novel technologies advance CTGF research in human cell systems?

Emerging technologies offer promising avenues to address current limitations and expand CTGF research capabilities in human cell systems. CRISPR/Cas9 genome editing enables precise manipulation of CTGF and interacting genes, allowing creation of knockout, knock-in, and domain-specific modifications in relevant cell types. Single-cell transcriptomics can reveal heterogeneity in CTGF responses across cell populations and identify previously unrecognized CTGF-responsive cell subsets. Proteomics approaches, particularly proximity labeling methods like BioID or APEX, can map CTGF's protein interaction networks in living cells, potentially uncovering novel binding partners beyond the established interactions with ER and extracellular matrix components . Advanced imaging techniques, including super-resolution microscopy, can visualize CTGF localization and trafficking within cells and tissues with unprecedented detail. For translational applications, patient-derived organoids or induced pluripotent stem cell (iPSC) models offer more physiologically relevant systems than traditional cell lines for studying CTGF functions in disease contexts. High-content screening platforms can accelerate the identification of compounds that modulate CTGF expression or activity, potentially leading to therapeutic applications as suggested by studies on blocking the MAS-CTGF-collagen pathway in heart failure .

What potential therapeutic applications might emerge from CTGF research in HEK systems?

Research on CTGF expression in HEK systems has revealed several promising avenues for therapeutic development across multiple disease areas. In fibrotic disorders, CTGF's role as a critical mediator of TGF-β2 signaling and ECM production suggests that targeting CTGF might alleviate pathological matrix accumulation without completely abolishing essential TGF-β functions . Studies showing that CTGF-specific siRNA inhibits TGF-β2-induced upregulation of fibronectin provide proof-of-concept for this approach . For cardiovascular applications, the identified MAS-CTGF-collagen pathway represents a potential target for pharmacological intervention in heart failure, as suggested by increased expression of MAS, CTGF, and collagen genes in failing human heart samples . The discovery that CTGF physically interacts with estrogen receptors and inhibits their transcriptional activity opens possibilities for modulating estrogen signaling in hormone-responsive cancers . Additionally, CTGF's involvement in integrin expression, particularly subunits αV and β1 , suggests potential applications in targeting cell adhesion and migration in metastatic disease. The ability to produce recombinant CTGF in HEK systems facilitates the development of neutralizing antibodies or decoy receptors that might block specific CTGF functions. As our understanding of domain-specific CTGF functions continues to grow, more targeted approaches may emerge that selectively inhibit pathological CTGF activities while preserving beneficial functions.