Heparan Sulfate Proteoglycans Research Articles

Neuronal Glypican4 promotes mossy fiber sprouting through the mTOR pathway after pilocarpine-induced status epilepticus in mice

In temporal lobe epilepsy (TLE), abnormal axon guidance and synapse formation lead to sprouting of mossy fibers in the hippocampus, which is one of the most consistent pathological findings in patients and animal models with TLE. Glypican 4 (Gpc4) belongs to the heparan sulfate proteoglycan family, which play an important role in axon guidance and excitatory synapse formation. However, the role of Gpc4 in the development of mossy fibers sprouting (MFS) and its underlying mechanism remain unknown. Using a pilocarpine-induced mice model of epilepsy, we showed that Gpc4 expression was significantly increased in the stratum granulosum of the dentate gyrus at 1 week after status epilepticus (SE). Using Gpc4 overexpression or Gpc4 shRNA lentivirus to regulate the Gpc4 level in the dentate gyrus, increased or decreased levels of netrin-1, SynI, PSD-95, and Timm score were observed in the dentate gyrus, indicating a crucial role of Gpc4 in modulating the development of functional MFS. The observed effects of Gpc4 on MFS were significantly antagonized when mice were treated with L-leucine or rapamycin, an agonist or antagonist of the mammalian target of rapamycin (mTOR) signal, respectively, demonstrating that mTOR pathway is an essential requirement for Gpc4-regulated MFS. Additionally, the attenuated spontaneous recurrent seizures (SRSs) were observed during chronic stage of the disease by suppressing the Gpc4 expression after SE. Altogether, our findings demonstrate a novel control of neuronal Gpc4 on the development of MFS through the mTOR pathway after pilocarpine-induced SE. Our results also strongly suggest that Gpc4 may serve as a promising target for antiepileptic studies.

An Atlas of Heparan Sulfate Proteoglycans in the Postnatal Rat Lens.

PurposeThe arrangement of lens cells is regulated by ocular growth factors. Although the effects of these inductive molecules on lens cell behavior (proliferation, survival, and fiber differentiation) are well-characterized, the precise mechanisms underlying the regulation of growth factor-mediated signaling in lens remains elusive. Increasing evidence highlights the importance of heparan sulfate proteoglycans (HSPGs) for the signaling regulation of growth factors; however, the identity of the different lens HSPGs and the specific roles they play in lens biology are still unknown.MethodsSemiquantitative real-time (RT)‐PCR and immunolabeling were used to characterize the spatial distribution of all known HSPG core proteins and their associated glycosaminoglycans (heparan and chondroitin sulfate) in the postnatal rat lens. Fibroblast growth factor (FGF)-2-treated lens epithelial explants, cultured in the presence of Surfen (an inhibitor of heparan sulfate [HS]-growth factor binding interactions) were used to investigate the requirement for HS in FGF-2-induced proliferation, fiber differentiation, and ERK1/2-signaling.ResultsThe lens expresses all HSPGs. These HSPGs are differentially localized to distinct functional regions of the lens. In vitro, inhibition of HS-sulfation with Surfen blocked FGF-2-mediated ERK1/2-signaling associated with lens epithelial cell proliferation and fiber differentiation, highlighting that these cellular processes are dependent on HS.ConclusionsThese findings support a requirement for HSPGs in FGF-2 driven lens cell proliferation and fiber differentiation. The identification of specific HSPG core proteins in key functional lens regions, and the divergent expression patterns of closely related HSPGs, suggests that different HSPGs may differentially regulate growth factor signaling networks leading to specific biological events involved in lens growth and maintenance.

Malaria parasite liver stages

Malaria parasite liver stages

DAL-1/4.1B promotes the uptake of exosomes in lung cancer cells via Heparan Sulfate Proteoglycan 2 (HSPG2).

DAL-1/4.1B is frequently absent in lung cancer tissues, which is significantly related to the occurrence and development of lung cancer. In this research, we found that DAL-1/4.1B affected the uptake of exosomes by lung cancer cells. When the expression of DAL-1/4.1B increased and decreased, the ability of exosome uptake enhanced and attenuated correspondingly. And we found that when cells were treated with different vesicles uptake inhibitors (chlorpromazine, methyl-β-cyclodextrin (MβCD), cytochalasin D, chloroquine and heparin) and heparinase (HSPE), only heparin and HSPE counteracted the uptake enhancement effect caused by DAL-1/4.1B. Therefore, we speculated that DAL-1/4.1B might promote the uptake of exosomes through the heparan sulfate proteoglycans (HSPGs) pathway. After screening the expression of HSPGs and HSPE in H292 cells, the expression of heparan sulfate proteoglycan 2 (HSPG2) increased with overexpression of DAL-1/4.1B and decreased with knockdown of DAL-1/4.1B. Meanwhile, exosome uptake decreased with HSPG2 knockdown in H292 and DAL-1/4.1B-overexpressing H292 cells. Moreover, knockdown of DAL-1/4.1B and HSPG2 in lung cancer A549 cells resulted in a similar decrease in exosome uptake, and the expression of HSPG2 was also decreased with DAL-1/4.1B knockdown. These results indicated that HSPG2 directly affected the uptake of exosomes, while DAL-1/4.1B positively affected the expression of HSPG2. Therefore, DAL-1/4.1B may promote cellular adhesion and inhibit migration in cancer cells.

The Heparanase Regulatory Network in Health and Disease.

The extracellular matrix (ECM) is a structural framework that has many important physiological functions which include maintaining tissue structure and integrity, serving as a barrier to invading pathogens, and acting as a reservoir for bioactive molecules. This cellular scaffold is made up of various types of macromolecules including heparan sulfate proteoglycans (HSPGs). HSPGs comprise a protein core linked to the complex glycosaminoglycan heparan sulfate (HS), the remodeling of which is important for many physiological processes such as wound healing as well as pathological processes including cancer metastasis. Turnover of HS is tightly regulated by a single enzyme capable of cleaving HS side chains: heparanase. Heparanase upregulation has been identified in many inflammatory diseases including atherosclerosis, fibrosis, and cancer, where it has been shown to play multiple roles in processes such as epithelial-mesenchymal transition, angiogenesis, and cancer metastasis. Heparanase expression and activity are tightly regulated. Understanding the regulation of heparanase and its downstream targets is attractive for the development of treatments for these diseases. This review provides a comprehensive overview of the regulators of heparanase as well as the enzyme’s downstream gene and protein targets, and implications for the development of new therapeutic strategies.

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants.

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

The Chemokine-Based Peptide, CXCL9(74-103), Inhibits Angiogenesis by Blocking Heparan Sulfate Proteoglycan-Mediated Signaling of Multiple Endothelial Growth Factors

Simple SummaryMajor angiogenic growth factors activate downstream signaling cascades by interacting with both receptor tyrosine kinases (RTKs) and cell surface proteoglycans, such as heparan sulfate proteoglycans (HSPGs). As current anti-angiogenesis regimens in cancer are often faced with resistance, alternative therapeutic strategies are highly needed. The aim of our study was to investigate the impact on angiogenic signaling when we interfered with growth factor-HSPG interactions using a CXCL9 chemokine-derived peptide with high affinity for HS.Growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) are important angiogenesis-mediating factors. They exert their effects not only through their respective receptor tyrosine kinases (RTKs), but they also require molecular pairing with heparan sulfate proteoglycans (HSPGs). Angiogenic growth factors and their signaling pathways are commonly targeted in current anti-angiogenic cancer therapies but have unfortunately insufficient impact on patient survival. Considering their obvious role in pathological angiogenesis, HS-targeting drugs have become an appealing new strategy. Therefore, we aimed to reduce angiogenesis through interference with growth factor-HS binding and downstream signaling using a CXCL9-derived peptide with a high affinity for glycosaminoglycans (GAGs), CXCL9(74-103). We showed that CXCL9(74-103) reduced EGF-, VEGF165- and FGF-2-mediated angiogenic processes in vitro, such as endothelial cell proliferation, chemotaxis, adhesion and sprouting, without exerting cell toxicity. CXCL9(74-103) interfered with growth factor signaling in diverse ways, e.g., by diminishing VEGF165 binding to HS and by direct association with FGF-2. The dependency of CXCL9(74-103) on HS for binding to HMVECs and for exerting its anti-angiogenic activity was also demonstrated. In vivo, CXCL9(74-103) attenuated neovascularization in the Matrigel plug assay, the corneal cauterization assay and in MDA-MB-231 breast cancer xenografts. Additionally, CXCL9(74-103) reduced vascular leakage in the retina of diabetic rats. In contrast, CXCL9(86-103), a peptide with low GAG affinity, showed no overall anti-angiogenic activity. Altogether, our results indicate that CXCL9(74-103) reduces angiogenesis by interfering with multiple HS-dependent growth factor signaling pathways.

Role of HSPGs in Systemic Bacterial Infections.

Heparan sulfate proteoglycans (HSPGs) are at the forefront of host-microbe interactions. Cell surface HSPGs are thought to promote infection as attachment and internalization receptors for many bacterial pathogens and as soluble inhibitors of host immunity when released from the cell surface by ectodomain shedding. However, the importance of HSPG-pathogen interactions in vivo has yet to be clearly established. Here we describe several representative methods to study the role of HSPGs in systemic bacterial infections, such as bacteremia and sepsis. The overall experimental strategy is to use mouse models to establish the physiological significance of HSPGs, to determine the identity of HSPGs that specifically promote infection, and to define key structural features of HSPGs that enhance bacterial virulence in systemic infections.

Isolation and Characterization of Heparan Sulfate Containing Amyloid Precursor Protein Degradation Products.

Numerous studies indicate that heparan sulfate proteoglycans (HSPGs) participate in a network of complex molecular events involving amyloid precursor protein (APP) processing and formation, oligomerization, intracellular targeting, clearance, and propagation of amyloid β in Alzheimer's disease (AD). A mutual functional interplay between recycling glypican-1 and APP processing has been demonstrated where the HS released from glypican-1 by a Cu/NO-ascorbate-dependent reaction forms a conjugate with APP degradation products and undergoes an endosome-nucleus-autophagosome co-trafficking. HS has been shown to display contradictory and dual effects in AD involving both prevention and promotion of amyloid β formation. It is therefore important to identify the source, detailed structural features as well as factors that favor formation of the neuroprotective forms of HS. Here, a method for isolation and identification of HS-containing APP degradation products has been described. The method is based on isolation of radiolabeled HS followed by identification of accompanying APP degradation products by SDS-PAGE and Western blotting.

Analyzing the Role of Heparan Sulfate Proteoglycans in Axon Guidance In Vivo in Zebrafish.

One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly wired to form functional circuits. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, less is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins.

Investigating the Roles of Heparan Sulfate Structures in Alpha-Synuclein Aggregation in Cell Culture Models.

Glycosaminoglycans (GAGs), belonging to a family of negatively charged linear polysaccharides, have been found in the cores of amyloid inclusions such as Lewy bodies, which are the central pathological features in Parkinson's disease (PD), a neurodegenerative disease. Lewy bodies/neurites are mostly composed of α-synuclein protein (α-syn) aggregates. Recent studies have shown that α-syn aggregates can propagate via neurons in a prion-like fashion by seeding the endogenous cellular α-syn. Various GAGs, especially heparan sulfate (HS), have been shown to be very critical in the aggregation of α-syn. HS chains of heparan sulfate proteoglycans (HSPGs) mediate the uptake of α-syn aggregates and help seed intracellular accumulation and further neuronal spread. Methods that inhibit the binding of these aggregates to HSPG have been shown to decrease the aggregate uptake and propagation. Here, we describe a cell-based assay to screen inhibitors of HS and α-syn interactions.

Generation of Drosophila Heparan Sulfate Mutant Cell Lines from Existing Fly Strains.

Genetic studies using a model organism, Drosophila melanogaster, have been contributing to elucidating the in vivo functions of heparan sulfate proteoglycans (HSPGs). On the other hand, biochemical analysis of Drosophila glycosaminoglycans (GAGs) has been limited, mainly due to the insufficient amount of the material obtained from the animal. Recently, a novel in vitro system has been developed by establishing mutant cell lines for heparan sulfate (HS)-modifying enzyme genes. Metabolic radiolabeling of GAGs allows us to assess uncharacterized features of Drosophila GAGs and the effects of the mutations on HS structures and function. The novel in vitro system will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.

Xyloside Derivatives as Molecular Tools to Selectively Inhibit Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects, making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner. Reversible and selective inhibition of GAG biosynthesis would allow us to examine the importance of endogenous GAGs to specific cellular, tissue, or organ systems. In this chapter, we describe the chemical synthesis and biological evaluation of xyloside derivatives as selective inhibitors of heparan sulfate and chondroitin/dermatan sulfate proteoglycan biosynthesis.

On the Selectivity of Heparan Sulfate Recognition by SARS-CoV-2 Spike Glycoprotein.

SARS-CoV-2 infects human cells through its surface spike glycoprotein (SgP), which relies on host cell surface heparan sulfate (HS) proteoglycans that facilitate interaction with the ACE2 receptor. Targeting this process could lead to inhibitors of early steps in viral entry. Screening a microarray of 24 HS oligosaccharides against recombinant S1 and receptor-binding domain (RBD) proteins led to identification of only eight sequences as potent antagonists; results that were supported by detailed dual-filter computational studies. Competitive studies using the HS microarray suggested almost equivalent importance of IdoA2S–GlcNS6S and GlcNS3S structures, which were supported by affinity studies. Exhaustive virtual screening on a library of >93 000 sequences led to a novel pharmacophore with at least two 3-O-sulfated GlcN residues that can engineer unique selectivity in recognizing the RBD. This work puts forward the key structural motif in HS that should lead to potent and selective HS or HS-like agents against SARS-CoV-2.

The Unifying Hypothesis of Alzheimer's Disease: Heparan Sulfate Proteoglycans/Glycosaminoglycans Are Key as First Hypothesized Over 30 Years Ago.

The updated “Unifying Hypothesis of Alzheimer’s disease” (AD) is described that links all the observed neuropathology in AD brain (i.e., plaques, tangles, and cerebrovascular amyloid deposits), as well as inflammation, genetic factors (involving ApoE), “AD-in-a-Dish” studies, beta-amyloid protein (Aβ) as a microbial peptide; and theories that bacteria, gut microflora, gingivitis and viruses all play a role in the cause of AD. The common link is the early accumulation of heparan sulfate proteoglycans (HSPGs) and heparan sulfate glycosaminoglycans (GAGs). HS GAG accumulation and/or decreased HS GAG degradation is postulated to be the key initiating event. HS GAGs and highly sulfated macromolecules induce Aβ 1–40 (but not 1–42) to form spherical congophilic maltese-cross star-like amyloid core deposits identical to those in the AD brain. Heparin/HS also induces tau protein to form paired helical filaments (PHFs). Increased sulfation and/or decreased degradation of HSPGs and HS GAGs that occur due to brain aging leads to the formation of plaques and tangles in AD brain. Knockout of HS genes markedly reduce the accumulation of Aβ fibrils in the brain demonstrating that HS GAGs are key. Bacteria and viruses all use cell surface HS GAGs for entry into cells, including SARS-CoV-2. Bacteria and viruses cause HS GAGs to rapidly increase to cause near-immediate aggregation of Aβ fibrils. “AD-in-a-dish” studies use “Matrigel” as the underlying scaffold that spontaneously causes plaque, and then tangle formation in a dish. Matrigel mostly contains large amounts of perlecan, the same specific HSPG implicated in AD and amyloid disorders. Mucopolysaccharidoses caused by lack of specific HS GAG enzymes lead to massive accumulation of HS in lysosomal compartments in neurons and contribute to cognitive impairment in children. Neurons full of HS demonstrate marked accumulation and fibrillization of Aβ, tau, α-synuclein, and prion protein (PrP) in mucopolysaccharidosis animal models demonstrating that HS GAG accumulation is a precursor to Aβ accumulation in neurons. Brain aging leads to changes in HSPGs, including newly identified splice variants leading to increased HS GAG sulfation in the AD brain. All of these events lead to the new “Unifying Hypothesis of Alzheimer’s disease” that further implicates HSPGs /HS GAGs as key (as first hypothesized by Snow and Wight in 1989).

Optimal Extracellular Matrix Niches for Neurogenesis: Identifying Glycosaminoglycan Chain Composition in the Subventricular Neurogenic Zone.

In the adult mammalian brain, new neurons are generated in a restricted region called the neurogenic niche, which refers to the specific regulatory microenvironment of neural stem cells (NSCs). Among the constituents of neurogenic niches, the extracellular matrix (ECM) has emerged as a key player in NSC maintenance, proliferation, and differentiation. In particular, heparan sulfate (HS) proteoglycans are capable of regulating various growth factor signaling pathways that influence neurogenesis. In this review, we summarize our current understanding of the ECM niche in the adult subventricular zone (SVZ), with a special focus on basement membrane (BM)-like structures called fractones, and discuss how fractones, particularly their composition of glycosaminoglycans (GAGs), may influence neurogenesis.

METHYLENE BLUE INHIBITS THE INTERACTION BETWEEN HEPARAN SULFATE AND SARS-COV-2 SPIKE PROTEIN; A REVIEW OF EVIDENCE FOR A HYPOTHESIS.

The addition of methylene blue to the standard treatment protocol has been shown to improve respiratory rate and oxygen saturation in COVID-19 patients, reducing morbidity and mortality. Evidence to date suggests that methylene blue inhibits protein-protein interactions between SARS-CoV-2 Spike protein and angiotensin-converting enzyme 2, which in turn inhibits the cell entry of SARS-CoV-2. However, the methylene blue dye-binding characteristics of sulfated glycosaminoglycans suggest additional inhibitory effects of the spike protein-heparan sulfate interaction. We hypothesize that the binding of cationic methylene blue neutralizes polyanionic heparan sulfate molecules on the host cell surface. As a consequence, electrostatic interactions between negatively charged heparan sulfate and the positively charged receptor binding domain of SARS-CoV-2 spike protein will be inhibited. Thus, methylene blue will exhibit a "shielding effect" on the heparan sulfate proteoglycans, inhibiting viral attachment to the cell surface. The proposed mechanism corroborates the possible broad-spectrum antiviral activity of methylene blue against multiple human coronaviruses that exploit the electrostatic interactions with sulfated glycosaminoglycans for virus entry. Methylene blue would exhibit the same anti-adhesive activity at the blood-brain-barrier and olfactory neuroepithelium, corroborating potential benefits in ameliorating post-COVID-19 neurological impairment. However, as cationic dyes can bind to both free glycosaminoglycans in circulation as well as proteoglycans attached to the cell surface, co-administration of intravenous heparin could possibly antagonize the proposed antiviral activity. This critical review focuses on empirical evidence to support the hypothesized heparan sulfate-dependent antiviral activity of MB.

Value of Glypican-3 as a Diagnostic and Prognostic Biomarker for Hepatocellular Carcinoma before and after Treatment. A Prospective Study

Background: Hepatocellular carcinoma is a highly prevalent tumor globally and the world's second leading cause of cancer-related deaths. Glypican-3, a heparan sulfate proteoglycan expressed on the surface of HCC cells, has emerged as a new molecule with a strong link to occurrence and progression of HCC. Objective: This study was done to determine the role of Glypican-3 in diagnosis of HCC and its prognostic value following different treatment modalities. Patients and methods: The study included thirty patients with liver cirrhosis and HCC on top, and thirty patients with liver cirrhosis without HCC. Standard laboratory investigations, abdominal ultrasound and triphasic computed tomography were done for all patients. Serum alpha fetoprotein and Glypican-3 were measured in all patients before and one month after different treatment modalities. Results: Glypican-3 was significantly higher in HCC group (2.28 ± 0.97) in comparison to cirrhosis group (0.56 ± 0.31) with P-value 1.1 ng/ml Glypican-3 has 93.3% sensitivity, 96.67% specificity, 96.6% PPV and 93.5% NPV for detection of HCC. Conclusion: Glypican-3 can be a valuable diagnostic marker for HCC diagnosis and prognosis after various treatment modalities and may be complementary to alpha fetoprotein increasing overall sensitivity of HCC detection.

Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS‐CoV‐2. Notably, neutralizing antibodies against SARS‐CoV‐2 isolated from COVID‐19 patients interfered with SARS‐CoV‐2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS‐CoV‐2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS‐CoV‐2, both DC subsets efficiently captured SARS‐CoV‐2 via heparan sulfate proteoglycans and transmitted the virus to ACE2‐positive cells. Notably, human primary nasal cells were infected by SARS‐CoV‐2, and infection was blocked by pre‐treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS‐CoV‐2 infection.

CD138 expression is a molecular signature but not a developmental requirement for RORγt+ NKT17 cells.

Invariant NKT (iNKT) cells are potent immunomodulatory cells that acquire effector function during their development in the thymus. IL-17–producing iNKT cells are commonly referred to as NKT17 cells, and they are unique among iNKT cells to express the heparan sulfate proteoglycan CD138 and the transcription factor RORγt. Whether and how CD138 and RORγt contribute to NKT17 cell differentiation, and whether there is an interplay between RORγt and CD138 expression to control iNKT lineage fate, remain mostly unknown. Here, we showed that CD138 expression was only associated with and not required for the differentiation and IL-17 production of NKT17 cells. Consequently, CD138-deficient mice still generated robust numbers of IL-17–producing RORγt+ NKT17 cells. Moreover, forced expression of RORγt significantly promoted the generation of thymic NKT17 cells, but did not induce CD138 expression on non-NKT17 cells. These results indicated that NKT17 cell generation and IL-17 production were driven by RORγt, employing mechanisms that were independent of CD138. Therefore, our study effectively dissociated CD138 expression from the RORγt-driven molecular pathway of NKT17 cell differentiation.