As discussed in a previous blog post, Plakophilin-4 (PKP4) is a recently discovered FLCN interactor that regulates RhoA signalling (Nahorski et al., 2012). A similar set of experiments by Medvetz et al. (2012) also found that PKP4 interacts with FLCN; that FLCN activates PKP4 expression and that FLCN loss leads to disordered cell colony formation. However, the two studies demonstrate opposing effects on RhoA signalling upon the loss of FLCN.
RhoA activity correlates with cell migration and metastasis (Liu et al., 2009). Therefore, both teams performed wound healing assays, comparing FLCN null and control cells. Nahorski et al. observed improved wound healing in FLCN null cells, consistent with RhoA activation; whereas Medvetz et al. observed impaired wound healing in FLCN null cells, consistent with a reduction in RhoA signalling.
Both studies investigated how cell-cell adhesion was affected by FLCN, and report contrasting results. Medvetz et al. grew UOK257 cells in hanging drops, and then sheared the resultant colonies. These colonies were more resistant to shearing than control colonies, and electron microscopy revealed that confluent FLCN-positive UOK257-2 cells lacked desmosomes. Taken together, these findings suggest that FLCN inhibits cell junction formation. In contrast, Nahorski et al. found that IMCD-3 cells expressing siRNAs directed against FLCN or PKP4 had a much lower trans-epithelial electrical resistance than control cells. Additionally, immunofluorescence staining showed Claudin‑1 and E-Cadherin to be mis‑localised in these cells, suggesting that cell junctions were not properly formed in cells lacking FLCN.
In order to investigate the role of FLCN on cell-cell adhesion in vivo, Medvetz et al. used keratin-14 Cre-recombinase (K14-Cre) to conditionally inactivate epidermal FLCN in mice. By three weeks post-partum, mice lacking epidermal FLCN were smaller than wild type littermates and displayed coarse, wavy fur; bald patches and delayed eye-opening. This phenotype resembles those seen in other cell-cell adhesion gene knockouts (Perez-Moreno et al., 2006) suggesting that this process is affected in these mice. On a cellular level, these mice showed hyper-proliferation of the epidermis. Inhibition of a downstream effector of the Rho signalling pathway, known as ROCK, increases keratinocyte proliferation (McMullan et al., 2003), suggesting that the epidermal phenotype seen in FLCN deficient mice is due to the inhibition of Rho signalling.
Although it seems that these studies are contradictory, it is worth noting that they used different cell lines. The Medvetz et al. experiments predominantly used UOK257 and UOK257-2 cells, which were isolated from a human BHD renal cell carcinoma; the Nahorski et al. experiments were carried out in a variety of cells lines, including thyroid carcinoma (FTC-133) cells and kidney adenocarcinoma (ACHN) cells. Additionally, cell confluence can affect Rho signalling (Zandy et al., 2007) and it is not known whether the confluence of the cells used to measure RhoA activity were the same in these two studies. However, it is plausible that the role of FLCN, and therefore also PKP4, is dependent on cell type and perhaps subject to modification by other proteins that are differentially expressed.
What is certain from these studies is that FLCN interacts with PKP4 to affect RhoA signalling. It will be interesting to see whether further research finds that Folliculin’s role in RhoA signalling is indeed context dependent, as has been reported for its role in mTOR signalling (Hudon et al., 2010).
- Hudon V, Sabourin S, Dydensborg AB, Kottis V, Ghazi A, Paquet M, Crosby K, Pomerleau V, Uetani N, & Pause A (2010). Renal tumour suppressor function of the Birt-Hogg-Dubé syndrome gene product folliculin. Journal of medical genetics, 47 (3), 182-9 PMID: 19843504
- Liu S, Goldstein RH, Scepansky EM, & Rosenblatt M (2009). Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone. Cancer research, 69 (22), 8742-51 PMID: 19887617
- McMullan R, Lax S, Robertson VH, Radford DJ, Broad S, Watt FM, Rowles A, Croft DR, Olson MF, & Hotchin NA (2003). Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway. Current biology : CB, 13 (24), 2185-9 PMID: 14680635
- Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H, & Henske EP (2012). Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion. PloS one, 7 (11) PMID: 23139756
- Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics PMID: 22965878
- Perez-Moreno M, Davis MA, Wong E, Pasolli HA, Reynolds AB, & Fuchs E (2006). p120-catenin mediates inflammatory responses in the skin. Cell, 124 (3), 631-44 PMID: 16469707
- Zandy NL, Playford M, & Pendergast AM (2007). Abl tyrosine kinases regulate cell-cell adhesion through Rho GTPases. Proceedings of the National Academy of Sciences of the United States of America, 104 (45), 17686-91 PMID: 17965237
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.