Recombinant Human Interleukin-20 receptor subunit alpha (IL20RA), partial (Active)

What receptor complexes does IL20RA form and with which cytokines does it interact?

IL20RA forms two distinct heterodimeric receptor complexes:

• Type I receptor complex: IL20RA/IL20RB heterodimer, which binds to IL-19, IL-20, and IL-24

• Type II receptor complex: IL22RA1/IL20RB heterodimer, which binds to IL-20 and IL-24

IL20RA belongs to the type II cytokine receptor family. Upon binding to its ligands (IL-19, IL-20, IL-24), IL20RA forms a functional heterodimeric receptor with IL20RB. Another IL20RA ligand, IL-26, requires IL20RA and IL10RB for signaling .

How is IL20RA expression regulated in normal tissues versus pathological conditions?

• In breast cancer: IL20RA is highly expressed and positively associated with SOX2 expression

• In colorectal cancer (CRC): IL20RA shows elevated expression compared to normal tissue

• In ovarian cancer: IL20RA expression decreases dramatically during peritoneal metastasis

• In inflammatory conditions: IL20RA expression is induced during remission in IBD

Experimental approaches to study IL20RA expression include immunohistochemical staining with scoring based on staining intensity and percentage of positive cells, western blot analysis, and TCGA dataset analysis .

What are the primary signaling pathways activated downstream of IL20RA?

IL20RA primarily signals through the JAK-STAT pathway, with distinct downstream effects:

• In breast cancer: IL20RA activates the JAK1-STAT3-SOX2 signaling pathway

• In osteoclast differentiation: IL20RA signaling activates the RANKL/TRAF6/NF-κB signaling pathway

• In intestinal inflammation: IL20RA activates STAT3 and suppresses interferon (IFN)-STAT2 signaling

Methods to investigate these signaling pathways include:

• Phosphorylation analysis by western blot

• Gain- and loss-of-function experiments using siRNA or shRNA

• Gene expression analysis by qRT-PCR and RNA-sequencing

• Co-immunoprecipitation for protein-protein interaction studies

How do IL20RA-mediated signaling pathways differ across tissue types?

IL20RA signaling exhibits tissue-specific effects through activation of different downstream mediators:

In breast cancer cells:

• Activates JAK1-STAT3-SOX2 pathway

• Increases expression of PD-L1

• Promotes stemness through upregulation of Sox2 and Oct4

In osteoclast precursors:

• Regulates RANKL-mediated osteoclastogenic downstream signal transduction

• Activates JNK, NF-κB, TRAF6, IκK, NFATc1, and p38 pathways

In intestinal epithelial cells:

• Induces STAT3 phosphorylation

• Suppresses IFN/STAT2 death signaling pathway

• Inhibits necroptotic cell death

In ovarian cancer cells:

• Triggers STAT3 phosphorylation

• Increases OAS1A expression

• Activates Caspase-1 and promotes IL-18 production by cleavage

When designing experiments to study IL20RA in different tissues, researchers should include appropriate tissue-specific controls and validate findings across multiple cell types .

How does IL20RA contribute to breast cancer progression and therapeutic resistance?

IL20RA plays a multifaceted role in breast cancer:

Stemness and tumor initiation:

• Increases side population (SP) and ALDH-bright proportions of breast cancer cells

• Enhances sphere formation ability

• Promotes expression of core stemness genes (Sox2, Oct4)

• Enhances tumor-initiating ability and lung metastasis in vivo

Chemoresistance:

• Increases resistance to chemotherapeutic agents

• Creates a tumor-favorable immune microenvironment

Immune modulation:

• Upregulates PD-L1 expression through JAK1-STAT3-SOX2 signaling

• Reduces recruitment of anti-cancer lymphocytes (CD8+ T cells, NK cells)

• Enhances the proportion of myeloid-derived suppressor cells

Therapeutic targeting:

• Combining anti-PD-L1 antibody with IL20RA-targeted delivery of STAT3 inhibitor (NP-Stattic-IL20RA) increases chemotherapeutic efficacy in breast cancer mouse models

These findings were established through multiple experimental approaches including flow cytometry, sphere formation assays, western blot analysis, and in vivo tumor models .

What is the role of IL20RA in colorectal cancer progression and metastasis?

In colorectal cancer (CRC), IL20RA functions as a promoter of tumor growth and metastasis:

Expression and regulation:

• IL20RA is upregulated in CRC tissues compared to normal tissues

• IL20RA is regulated by super-enhancers in CRC cells

Functional impact:

• Knockdown of IL20RA in LoVo cells significantly reduces cell growth, migration, and invasion

• IL20RA knockdown suppresses the expression of EMT pathway markers Snail and Slug

• Unlike other cancer types, IL20RA appears to function in a ligand-independent manner in CRC, as adding IL-20 did not alter CRC cell growth

Experimental approaches:

• shRNA-mediated knockdown of IL20RA

• Cell viability assays using Cell Counting Kit 8

• Migration and invasion assays

• Western blot analysis of EMT markers

These findings suggest IL20RA as a potential therapeutic target in CRC, with mechanisms distinct from its role in other cancer types .

What is the paradoxical role of IL20RA in ovarian cancer metastasis?

Contrary to its pro-oncogenic role in breast and colorectal cancers, IL20RA functions as a metastasis suppressor in ovarian cancer:

Expression pattern:

• IL20RA expression decreases dramatically in ovarian cancer patients during peritoneal metastasis

Anti-metastatic functions:

• Reconstitution of IL20RA in highly metastatic ovarian cancer cells suppresses transcoelomic metastasis

• IL20RA mediates a crosstalk between ovarian cancer cells and peritoneal mesothelial cells

Molecular mechanism:

• Ovarian cancer cells in the peritoneal cavity induce mesothelial cells to express IL-20 and IL-24

• These cytokines activate IL20RA downstream signaling in ovarian cancer cells

• IL20RA signaling triggers STAT3 phosphorylation and increases OAS1A and activated Caspase-1

• This leads to production of mature IL-18

• IL-18 ultimately polarizes macrophages into the M1-like subtype that can clear cancer cells

Experimental validation:

• CRISPR/Cas9 screen identified IL20RA as a key factor preventing transcoelomic metastasis

• In vivo models confirmed the suppressive role of IL20RA in peritoneal dissemination

• Knockdown of IL20RB (heterodimer partner) abolished the suppressive effects of IL20RA

This dual role of IL20RA as both oncogenic and tumor-suppressive in different cancer types highlights the context-dependent nature of cytokine signaling in cancer biology .

How does the IL20RA signaling pathway influence bone homeostasis and osteoclastogenesis?

IL20RA signaling plays a critical role in regulating bone homeostasis through effects on osteoclast differentiation and function:

Dose-dependent effects of IL-20:

• Low concentration (20 ng/mL): Promotes BMM proliferation, increases TRAP-positive osteoclasts, enhances bone resorption

• High concentration (>100 ng/mL): Inhibits BMM proliferation and osteoclastogenesis

Molecular mechanisms:

• IL-20 at 20 ng/mL upregulates BMM proliferation signaling factors (GRB2, ERK, NF-κB)

• Modulates expression of osteoclast-specific and bone resorption functional proteins (TRAP, CTSK, MMP-9)

• During early osteoclast differentiation, low IL-20 concentration upregulates RANK and CTSK expression

• IL-20 regulates RANKL-mediated osteoclastogenic downstream signaling through activation of JNK, NF-κB, TRAF6, IκK, NFATc1, and p38 pathways

Receptor specificity:

• IL20RB is essential for IL-20's effects on osteoclastogenesis

• When IL20RB is knocked down, IL-20 does not influence osteoclast differentiation

Indirect regulation through stromal cells:

• IL-20 induces BMSCs to regulate OPG and RANKL expression

• This affects osteoclastogenesis through the OPG/RANKL/RANK axis

These findings suggest potential therapeutic applications for IL20RA pathway modulators in bone loss diseases and osteoporosis .

What is the role of IL20RA in intestinal inflammation and colitis?

IL20RA plays a protective role in intestinal inflammation:

Clinical relevance in IBD:

• IL-20 levels are induced during remission in IBD

• IL-20 levels are significantly higher in anti-TNF responders versus non-responders

• IL20RA and IL20RB are expressed on intestinal epithelial cells (IECs) from IBD patients

Experimental evidence:

• Il20^-/-^, Il20ra^-/-^, and Il20rb^-/-^ mice show increased susceptibility to experimental DSS-induced colitis

• IECs are the main producers of IL-20 in IBD and during mucosal healing

Molecular mechanism:

• IL-20 activates STAT3 and suppresses interferon (IFN)-STAT2 signaling in IECs

• IL-20 deficiency is associated with increased IFN/STAT2 activity

• IL-20 blocks IFN/STAT2-induced necroptotic cell death in IEC-derived organoids

• Stat2^ΔIEC^ mice (lacking STAT2 in IECs) show reduced susceptibility to experimental colitis

• Administration of IL-20 suppresses colitis activity in wildtype animals

Experimental approaches:

• In vivo imaging and high-resolution mini-endoscopy

• Histological assessment of intestinal inflammation

• RNA-Seq and Gene Ontology analysis

• RNAScope for spatial gene expression

• 3D organoid models from intestinal epithelial cells

• Co-immunoprecipitation and confocal microscopy

These findings indicate potential new therapeutic approaches for IBD through modulation of the IL-20/IL20RA pathway .

What are the optimal techniques for studying IL20RA protein interactions and receptor complex formation?

Several complementary techniques are effective for studying IL20RA protein interactions:

Proximity Ligation Assay (PLA):

• The Duolink PLA system can detect IL20RA interactions with IL20RB or IL22RA1

• This technique detects proteins in close proximity (<40 nm)

• Uses primary antibodies raised in different host species (e.g., goat anti-IL20RB with mouse anti-IL20RA)

• Interaction is visualized through rolling-circle amplification and fluorescent oligonucleotide hybridization

• Particularly useful for detecting endogenous protein interactions in situ

Co-immunoprecipitation:

• Effective for studying IL20RA interactions with signaling components like STAT3

• Can detect protein complexes formed after cytokine stimulation

• Western blot analysis following co-IP confirms specific interactions

Western Blot Analysis:

• For evaluating IL20RA expression levels and activation of downstream signaling

• Can detect phosphorylation of STAT proteins (p-STAT3, p-STAT1)

• Useful for time-course experiments after cytokine stimulation

Recombinant Receptor Binding Assays:

• Using purified recombinant IL20RA with potential binding partners

• Can determine binding affinities and kinetics

• Useful for screening potential therapeutic modulators

Fluorescence Resonance Energy Transfer (FRET):

• For studying real-time receptor complex formation in living cells

• Requires fluorescent protein tagging of IL20RA and its binding partners

The choice of method depends on the specific research question, with PLA being particularly valuable for detecting endogenous protein interactions in their native cellular context .

What are the recommended approaches for knockdown and overexpression studies of IL20RA?

Several effective approaches for IL20RA genetic manipulation have been documented:

Knockdown approaches:

• shRNA-mediated knockdown:

  • Using pLV-H1-shIL20RA-puro plasmid (for stable knockdown)

  • Sequences specifically targeting IL20RA silencing:

    • shIL20RA#1: GCTATTCCATCTACCGATA

    • shIL20RA#2: GCCCGCAAACGTTACAGTA

  • Selection with puromycin to obtain stable polyclonal cell lines

• siRNA-mediated knockdown:

  • For transient knockdown experiments

  • Effective in bone marrow-derived mononuclear cells and other primary cells

  • Verify knockdown efficiency by qRT-PCR and western blotting

Overexpression approaches:

• Lentiviral-mediated overexpression:

  • Insert human IL20RA or mouse Il20ra into pLV-EF1α-MCS-IRES-Bsd plasmid

  • Co-transfect with packaging plasmids (psPAX2 and pMD2.G) into HEK293T cells

  • Select stable cell lines using blasticidin

• Plasmid transient transfection:

  • For short-term expression studies

  • Particularly useful in cells that are difficult to transduce with lentivirus

Validation methods:

• qRT-PCR to confirm mRNA levels

• Western blot to verify protein expression

• Functional assays to confirm biological effects of manipulation

Experimental considerations:

• Include appropriate vector controls (empty plasmid)

• Use multiple independent shRNA/siRNA sequences to rule out off-target effects

• Consider species-specific constructs when working with different model systems

• For cancer studies, validate findings in multiple cell lines to ensure generalizability

How can IL20RA-targeted therapies be developed for cancer treatment?

Development of IL20RA-targeted therapies requires a multifaceted approach:

Targeting strategies based on cancer type:

• Breast cancer: Inhibit IL20RA signaling to reduce stemness and immune evasion

• Colorectal cancer: Suppress IL20RA expression to inhibit EMT and metastasis

• Ovarian cancer: Enhance IL20RA signaling to promote anti-tumor immune responses

Therapeutic delivery systems:

• IL20RA-targeted liposomal nanoparticles:

  • NP-Stattic-IL20RA (nanoparticles encapsulating STAT3 inhibitor stattic with IL20RA targeting)

  • Enhanced drug delivery to IL20RA-expressing cancer cells

  • Demonstrated efficacy in breast cancer mouse models when combined with anti-PD-L1 antibody

Combination therapy approaches:

• Anti-PD-L1 antibody + IL20RA-targeted STAT3 inhibition + chemotherapy

• This triple combination showed superior efficacy in breast cancer models

• The rationale is targeting multiple aspects: immune checkpoint, IL20RA-mediated stemness, and cancer cell proliferation

Patient stratification considerations:

• IL20RA expression levels in tumors could serve as biomarkers for patient selection

• High IL20RA expression in breast cancer correlates with stemness features and SOX2 expression

• Tumor IL20RA expression could predict response to targeted therapies

Challenges to overcome:

• Context-dependent functions of IL20RA in different cancers

• Potential side effects on bone homeostasis and inflammatory responses

• Developing specific targeting strategies that avoid disrupting beneficial IL20RA functions

What are the current contradictions or knowledge gaps in IL20RA research?

Several significant contradictions and knowledge gaps exist in IL20RA research:

Opposing roles in different cancer types:

• Pro-tumorigenic in breast and colorectal cancers

• Anti-metastatic in ovarian cancer

• Research gap: Molecular determinants of these context-dependent functions

Dose-dependent effects of IL-20:

• Low concentrations (20 ng/mL) promote osteoclastogenesis

• High concentrations (>100 ng/mL) inhibit osteoclastogenesis

• Knowledge gap: Mechanisms behind this biphasic response and therapeutic implications

Ligand-dependent versus ligand-independent functions:

• IL20RA functions appear ligand-dependent in breast cancer and bone cells

• In colorectal cancer, IL20RA functions in a ligand-independent manner

• Research need: Characterization of ligand-independent signaling mechanisms

Heterodimeric receptor complexes:

• IL20RA can form complexes with both IL20RB and IL22RA1

• Knowledge gap: How these different receptor complexes regulate distinct biological processes

Tissue-specific expression patterns:

• Primarily co-expressed with IL20RB in skin and testis under normal conditions

• Expressed in various tumor types and immune cells in pathological conditions

• Research opportunity: Comprehensive tissue atlas of IL20RA expression in health and disease

Therapeutic targeting challenges:

• Beneficial in cancer versus potential disruption of normal physiological functions

• Research need: Development of tissue-specific targeting approaches

Addressing these contradictions requires integrative approaches combining multi-omics data, advanced imaging techniques, and sophisticated in vivo models .

How can single-cell approaches enhance our understanding of IL20RA biology?

Single-cell technologies offer powerful approaches to address complex questions in IL20RA research:

Single-cell RNA sequencing applications:

• Revealing cell type-specific expression patterns of IL20RA and its heterodimeric partners

• Identifying rare cell populations with unique IL20RA-dependent gene signatures

• Tracking dynamic changes in IL20RA signaling during disease progression

• Uncovering heterogeneity in IL20RA-responsive cells within tumors or inflamed tissues

Spatial transcriptomics integration:

• Mapping IL20RA expression in the spatial context of tissues

• Identifying microenvironmental factors influencing IL20RA function

• Correlating IL20RA expression with immune cell infiltration patterns

Single-cell protein analysis:

• Cytometry by time-of-flight (CyTOF) to simultaneously measure IL20RA with dozens of other proteins

• Single-cell western blotting to detect activation of IL20RA-downstream pathways

• Proximity ligation assays at single-cell resolution to identify receptor complex formation

Functional single-cell approaches:

• CRISPR-based single-cell perturbation screens targeting IL20RA pathway components

• Tracking IL20RA-dependent cell fate decisions in real-time

• Correlating single-cell IL20RA signaling with functional outcomes like migration or cytokine production

Data integration challenges:

• Computational approaches to integrate single-cell multi-omics data

• Machine learning algorithms to identify IL20RA-dependent cellular states

• Network analysis to place IL20RA in the context of broader signaling pathways