Filamins are large dimeric F-actin cross-linking proteins, crucial for the mechanosensitive properties of a number of cell types. Due to their interaction with a variety of different proteins, they exert important regulatory functions. However, in the human testis the role of filamins has been insufficiently explored. Immunohistochemical staining of human testis samples identified filamin A (FLNA) in spermatogonia and peritubular myoid cells. Investigation of different testicular tumor samples indicated that seminoma also express FLNA. Moreover, mass spectrometric analyses identified FLNA as one of the most abundant proteins in human seminoma TCam-2 cells. We therefore focused on FLNA in TCam-2 cells, and identified by co-immunoprecipitation LAD1, RUVBL1 and DAZAP1, in addition to several cytoskeletal proteins, as interactors of FLNA. To study the role of FLNA in TCam-2 cells, we generated FLNA-deficient cells using the CRISPR/Cas9 system. Loss of FLNA causes an irregular arrangement of the actin cytoskeleton and mechanical instability, impaired adhesive properties and disturbed migratory behavior. Furthermore, transcriptional activity of typical stem cell factors is increased in the absence of FLNA. In summary, our data suggest that FLNA is crucially involved in balancing stem cell characteristics and invasive properties in human seminoma cells and possibly human testicular germ cells.

The cation channel TRPV2 is known to be expressed by murine macrophages and is crucially involved in their functionality. Macrophages are frequent cells of the mouse testis, an immune-privileged and steroid-producing organ. TRPV2 expression by testicular macrophages and possible changes associated with age or inflammation have not been investigated yet. Therefore, we studied testes of young adult and old wild-type (WT) and AROM⁺ mice, i.e., transgenic mice overexpressing aromatase. In these animals, inflammatory changes are described in the testis, involving active macrophages, which increase with age. This is associated with impaired spermatogenesis and therefore AROM⁺ mice are a model for male infertility associated with sterile inflammation. In WT animals, testicular TRPV2 expression was mapped to interstitial CD206⁺ and peritubular MHC II⁺ macrophages, with higher levels in CD206⁺ cells. Expression levels of TRPV2 and most macrophage markers did not increase significantly in old mice, with the exception of CD206. As the number of TRPV2⁺ testicular macrophages was relatively small, their possible involvement in testicular functions and in aging in WT mice remains to be further studied. In AROM⁺ testis, TRPV2 was readily detected and levels increased significantly with age, together with macrophage markers and TNF-α. TRPV2 co-localized with F4/80 in macrophages and further studies showed that TRPV2 is mainly expressed by unusual CD206⁺MHC II⁺ macrophages, arising in the testis of these animals. Rescue experiments (aromatase inhibitor treatment and crossing with ERαKO mice) restored the testicular phenotype and also abolished the elevated expression of TRPV2, macrophage and inflammation markers. This suggests that TRPV2⁺ macrophages of the testis are part of an inflammatory cascade initiated by an altered sex hormone balance in AROM⁺ mice. The changes in testis are distinct from the described alterations in other organs of AROM⁺, such as prostate and spleen. When we monitored TRPV2 levels in another immune-privileged organ, namely the brain, we found that levels of TRPV2 were not elevated in AROM⁺ and remained stable during aging. In the adrenal, which similar to the testis produces steroids, we found slight, albeit not significant increases in TRPV2 in both AROM⁺ and WT mice, which were associated with age. Thus, the changes in the testis are specific for this organ.

Background: The transient receptor potential vanilloid 2 (TRPV2) channel is known to have strong species-dependent activation modes and divers functions. Based on previous results, which showed expression by human ovarian granulosa cells, w e studied human TRPV2 in human granulosa cell tumors (GCTs) and derived cells (KGN cells). GCTs are rare ovarian tumors, for which neither specific therapies, nor adequate markers are available. Methods: We analyzed primary GCTs, including a panel of 63 GCT samples, and KGN cells. We performed immunohisto-/cytochemistry, RT-PCR, Western blotting and measurements of intracellular Ca ²⁺ levels. We studied consequences of CRISPR/Cas9-based deletion of TRPV2 on cell proliferation, migration and macropinocytotic behavior, examined changes in steroid hormone production, and determined corresponding alterations of the proteome of these cells. Results: We found that GCTs express TRPV2 to a large percentage (95%). To examine roles of TRPV2, we turned to the human GCT-derived KGN cell line. CRISPR/Cas9-based deletion of TRPV2 resulted in larger cell size, increases in proliferation, migration and macropinocytotic behavior, changes in steroid production, and corresponding alterations of the proteome of these cells. Deletion of TRPV2 also significantly reduced susceptibility to cell death, which was induced within hours by cannabidiol (CBD), a preferred ligand of TRPV2, in a concentration- and time-dependent manner. However, cell death was not completely abolished and the analysis of the TRPV2-interactome suggested the voltage-dependent anion channel 1 (VDAC1) as a further target for CBD. VDAC1, as part of a cascade involving formation and persistent opening of the mitochondrial permeability transition pore (mPTP), is linked to cell death. A blocker of mPTP formation, cyclosporin A, significantly decreased the vulnerability of KGN cells to CBD-induced cell death. TRPV2-depleted KGN cells treated with cyclosporin A became almost completely insensitive to the effects of CBD. Conclusions: The results reveal a role of TRPV2 in GCT cells. Thus, CBD causes cell death in KGN cells via direct TRPV2 activation and via interaction with VDAC1. Expression of TRPV2 may thus be a novel marker to distinguish subtypes of GCTs. Furthermore, TRPV2 represents a novel drug target.