Recombinant Rat Transmembrane protein 106A (Tmem106a)

What is Tmem106a and what is its molecular structure?

Tmem106a is a type II transmembrane protein that is highly conserved across species. Structurally, mouse Tmem106a consists of 261 amino acids with an isoelectric point of 7.04 and features a conserved transmembrane domain (amino acids 93-115) . The gene is located on chromosome 11 in mice and contains nine exons and eight introns, with a full-length cDNA of 2301 base pairs . Bioinformatic analyses using transmembrane prediction tools (TMHMM-2.0) confirm its classification as a type II transmembrane protein . The protein is constitutively expressed in macrophages and plays significant roles in inflammatory regulation .

What is the tissue expression profile of Tmem106a?

Tmem106a exhibits tissue-specific expression patterns. High levels of Tmem106a mRNA are detected in lung, kidney, intestine, and lymphoid node tissues in mice . At the protein level, Western blot analysis has confirmed consistent expression in these tissues . Within the immune system, Tmem106a is highly expressed in myeloid cells, especially macrophages, as indicated by quantitative real-time PCR (qRT-PCR) analyses . Cell line studies have shown that Tmem106a protein is expressed in RAW264.7, NIH-3T3, P815, THP-1, and mouse peritoneal macrophages, but not in Sp2/0, HeLa, or MDA231 cells .

How does Tmem106a affect inflammatory responses?

Tmem106a plays a complex role in inflammatory regulation, with seemingly contradictory functions reported in different experimental settings. When knocked out, Tmem106a enhances inflammatory responses to lipopolysaccharide (LPS) stimulation, suggesting it normally functions as a negative regulator of inflammation . Tmem106a knockout mice display increased sensitivity to LPS-induced septic shock compared to wild-type counterparts, exhibiting elevated levels of pro-inflammatory cytokines like TNF-α, IL-6, and IFN-β in serum . Additionally, these knockout mice show more severe tissue damage, particularly in the lungs, with high inflammatory cell infiltration, hemorrhage, and interstitial pneumonitis after LPS challenge .

Contrastingly, direct activation of Tmem106a using anti-Tmem106a antibodies promotes inflammatory responses in macrophages, suggesting a pro-inflammatory role under certain conditions . This apparent contradiction highlights the context-dependent nature of Tmem106a function in inflammatory regulation.

What role does Tmem106a play in macrophage polarization?

Tmem106a significantly influences macrophage polarization. Activation of Tmem106a by anti-Tmem106a antibodies upregulates the expression of activation markers including CD80, CD86, CD69, and MHC II on macrophages . This activation induces the release of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, CCL2, and nitric oxide (NO), but not the anti-inflammatory cytokine IL-10 .

Notably, anti-Tmem106a treatment significantly increases inducible nitric oxide synthase (iNOS) production and STAT1 phosphorylation without affecting ARGINASE-1 or STAT6 phosphorylation . This cytokine profile and activation pattern indicates that Tmem106a activation polarizes macrophages toward an M1-like (pro-inflammatory) phenotype . Conversely, Tmem106a ablation also enhances the expression of M1 markers upon LPS stimulation, suggesting a complex regulatory role in macrophage polarization that may differ depending on the stimulus .

What are established methods for generating Tmem106a knockout models?

Researchers have successfully generated Tmem106a knockout mouse models using CRISPR/Cas9 genome editing. The specific approach involves:

• Guide RNA design: Using guide RNA (5′-GCTCACCTCTCGGAAGGATG-3′) targeting close to the start codon in exon 3 of mouse Tmem106a .

• Embryo injection: C57BL/6J × FVB/N mouse embryos were injected with gRNAs and Cas9 mRNA .

• Confirmation and breeding: Editing was confirmed by sequencing PCR products from genomic DNA. Offspring from founder containing 148 base pairs (bp) deletion were back-crossed to a C57/BL6 background for 10 generations .

• Genotyping protocol: Δ148 bp KO mice genotyping was performed by PCR using oligonucleotides 5′-TTCACTTGCAGAAATCCCTTAAA-3′ and 5′-GCCAGCCTGAGACTGCATAC-3′, yielding wild-type allele (577 bp) and mutant allele (429 bp) .

What methods are effective for Tmem106a knockdown in cell lines?

For stable Tmem106a knockdown in cell lines, researchers have successfully employed lentiviral shRNA approaches:

• shRNA design: The sequence 5′-GCTCAACACGACGAATGTCCT-3′ specifically targeting Tmem106a mRNA has proven effective when constructed into the transfer plasmid pLVX-shRNA1 .

• Lentivirus production: HEK 293T cells are transfected with 10 μg transfer plasmid, 6 μg packaging plasmid, and 4 μg envelope plasmid. Recombinant lentiviruses are harvested and filtered 72 hours post-transfection .

• Cell infection and selection: RAW264.7 cells infected with the lentivirus are selected under pressure of 5 μg/ml puromycin .

For transient knockdown, siRNA approaches have also been validated:

• siRNA transfection in peritoneal macrophages for 24 hours before treatment with control IgG, anti-Tmem106a, or LPS has shown effective knockdown .

• Two validated siRNAs (siTmem106a-1 and siTmem106a-2) have demonstrated efficacy in reducing Tmem106a expression in mouse peritoneal macrophages .

What signaling pathways are modulated by Tmem106a in inflammatory responses?

Tmem106a regulates inflammatory responses through several key signaling pathways:

• MAPK Pathway: Anti-Tmem106a stimulation increases phosphorylation of ERK-1/2, JNK, and p38 MAPK . Similarly, enhanced MAPK signaling is observed in LPS-induced inflammatory responses in Tmem106a-deficient macrophages .

• NF-κB Signaling: Anti-Tmem106a treatment increases phosphorylation of NF-κB p65 and IKKα/β, and promotes nuclear translocation of the cytosolic NF-κB p65 subunit . Elevated NF-κB signaling is also observed in Tmem106a knockout macrophages upon LPS stimulation .

• STAT Signaling: Anti-Tmem106a significantly increases phosphorylation of STAT1 without affecting STAT6 phosphorylation, consistent with M1 macrophage polarization .

These findings suggest that Tmem106a influences inflammatory responses through the coordinated regulation of multiple signaling cascades, potentially acting as both an activator (when directly stimulated) and a negative regulator (as observed in knockout models) in different contexts.

How is Tmem106a expression regulated during inflammation?

Tmem106a expression is dynamically regulated during inflammatory responses. Upon LPS stimulation, Tmem106a levels increase significantly in macrophages, as demonstrated by both qRT-PCR and flow cytometry analyses . This upregulation is observed in mouse bone marrow-derived macrophages (mBMDMs) at the mRNA level and in PMA-stimulated THP-1 cells at the protein level .

Specifically, treatment with LPS (100 ng/ml) enhances the protein expression of Tmem106a in PMA-stimulated THP-1 cells . This upregulation during inflammatory stimulation suggests that Tmem106a may function as part of a feedback mechanism to regulate the inflammatory response. Additionally, elevated Tmem106a levels have been observed in peripheral monocytes of patients with sepsis, indicating clinical relevance of its inflammatory regulation .

What is the role of Tmem106a in sepsis models?

Tmem106a plays a protective role in experimental sepsis models. Tmem106a knockout mice exhibit increased sensitivity to LPS-induced septic shock compared to wild-type mice . In the cecal ligation and puncture (CLP) model of polymicrobial sepsis, Tmem106a-deficient mice show dramatically reduced survival rates compared to wild-type counterparts. At 40 hours after CLP, approximately 50% of Tmem106a knockout mice died, while all wild-type mice survived . After 7 days, only 20% of Tmem106a knockout mice remained alive, compared to 50% of wild-type mice .

This increased susceptibility correlates with higher serum levels of pro-inflammatory cytokines (TNF and IL-6) and more severe lung injury in Tmem106a knockout mice at 12 hours after CLP . These findings suggest that Tmem106a functions as a negative regulator of inflammatory responses during sepsis, protecting against excessive inflammation and tissue damage.

What evidence exists for Tmem106a involvement in cancer?

Emerging evidence indicates that Tmem106a expression is dysregulated in several cancer types, particularly hepatocellular carcinoma (HCC). Tmem106a is markedly downregulated in HCC compared to normal liver tissue . This downregulation appears to be mediated by tumor-specific DNA methylation, which is frequently observed in tumor tissues from HCC patients .

Research has established a significant relationship between Tmem106a methylation and downregulation of protein expression as demonstrated by immunohistochemistry and pyrosequencing analyses . Receiver operating characteristic (ROC) curve analysis reveals that methylation of Tmem106a in tumor samples differs significantly from that in non-malignant adjacent tissues of HCC patients .

Importantly, HCC patients with Tmem106a hypermethylation have been found to have a poor clinical prognosis . These findings suggest that Tmem106a may function as a tumor suppressor in HCC, with epigenetic silencing contributing to disease progression.

How can bone marrow chimeras be used to study cell-specific roles of Tmem106a?

Bone marrow chimera experiments provide valuable insights into the cell-specific functions of Tmem106a, particularly in distinguishing between its roles in hematopoietic versus non-hematopoietic cells. The established protocol involves:

• Lethal irradiation of wild-type recipient mice to ablate endogenous bone marrow .

• Reconstitution with either Tmem106a-knockout or wild-type bone marrow cells .

• Challenge with LPS to assess inflammatory responses.

Using this approach, researchers have demonstrated that chimeric mice reconstituted with Tmem106a-deficient bone marrow (Tmem106a−/− → Tmem106a+/+) produce higher levels of pro-inflammatory cytokines (TNF, IL-6, and IFN-β) than those reconstituted with wild-type bone marrow following LPS challenge . This suggests that the inflammatory phenotype observed in Tmem106a knockout mice is primarily mediated by hematopoietic cells, particularly macrophages.

What methods are recommended for analyzing Tmem106a-mediated cytokine production?

For comprehensive analysis of Tmem106a-mediated cytokine production, researchers should employ the following methodologies:

• Multiplex cytokine assays: The LEGENDplex™ mouse proinflammatory chemokine panel (BioLegend, 740451) has been successfully used to measure multiple cytokines including IL-6, TNF-α, and IFN-β in both serum and cell culture supernatants .

• ELISA: Standard ELISA kits for specific cytokines (TNF-α, IL-1β, IL-6, CCL2, IL-10) can effectively quantify individual cytokine levels in culture supernatants from Tmem106a-activated or Tmem106a-deficient macrophages .

• Cell sources: Both primary cells (thioglycollate-elicited or non-elicited peritoneal macrophages) and cell lines (RAW264.7) can be used as experimental models, with primary cells typically providing more physiologically relevant results .

• Experimental design: Comparative analysis between anti-Tmem106a treatment (10 μg/mL), isotype control IgG (10 μg/mL), and LPS (5 μg/mL) provides comprehensive insights into Tmem106a-specific effects versus general inflammatory activation .