RhoH/TTF is a hematopoietic member of the Rho GTPase family and was shown to be expressed at very high levels in thymus, less abundant in spleen, and least abundant in the BM (Li et al., 2002). The highest RhoH expression is detected in lymphocytes, while hemato- poietic progenitor cells (HPCs), myeloid lineages, including neutrophils and in vitro BM- derived mast cells, (Li et al., 2002; Gu et al., 2005a) display lower expression. In erythroid cell lines RhoH is expressed at the lowest levels (Lahousse et al., 2004). RhoH seems not to be expressed in non-hematopoietic tissues.
The RhoH gene is located on chromosome 4 and 5 in humans and mice, respectively. The RhoH gene was first identified as a fusion transcript of LAZ3/BCL6 (B cell lymphoma 6) oncogene as a result of a t(3;4)(q27;p11-13) translocation in the B cell non-Hodgkin’s lymphoma (NHL) cell line and in other NHL cases (Dallery et al., 1995; Preudhomme et al.,
2000). LAZ3/BCL6 gene encodes a sequence-specific zinc finger transcriptional inhibitor (Albagli et al., 1995). It has been suggested that LAZ3/BCL6 is important for the repression of genes involved in the control of lymphocyte activation, differentiation, and apoptosis, and for the formation of germinal centre B cells, where its downregulation is further necessary for B cells to exit the germinal centres (Pasqualucci et al., 2003; Ohno, 2006). Often, LAZ3/BCL6 remains constitutively active in lymphomas. With respect to the RhoH LAZ3/BCL6 rearrangement, Preudhomme et al., 2000 reported two fusion products, a RhoH- LAZ3/BCL6 and a LAZ3/BCL6-RhoH, placing each gene under the control of the partner gene promoter. This exchange suggests a deregulated expression of both the LAZ3/BCL6 and the RhoH gene. In multiple myeloma, which develops from malignant, differentiated B lymphocytes accumulating in the BM, another RhoH translocation t(4;14)(p13;q32) was isolated, involving the IgH gene. The transposition of RhoH into the Ig locus conceivably also results in the deregulation of the RhoH gene expression (Preudhomme et al., 2000).
Recently, the RhoH gene along with three other genes (PIM1, MYC and PAX5) was discovered to be hypermutated in 46% of the diffuse large B cell lymphomas (DLBCLs), the most typical subtype of NHL (Pasqualucci et al., 2001). Mutations were also reported to occur in other subtypes of B cell NHL (Gaidano et al., 2003; Deutsch et al., 2007; Bödor et al., 2005; Dijkman et al., 2006). In contrast, RhoH gene is rarely affected by mutations in Hodgkin’s lymphoma (HL) (Liso et al., 2006).
The mutations are introduced by the aberrant somatic hypermutation (SHM) mechanism. Normally, this mechanism selectively targets the variable regions of Ig genes of the antigen- activated B cells which undergo clonal expansion in germinal centres during humoral immunity response, and inserts nucleotide substitutions to generate variants of Igs with higher affinity for antigen. The SHM is not only restricted to the Ig loci, but brings in mutations also into BCL6, CD95/Fas, CD79a and CD79b genes (Peng et al., 1999; Shen et al., 1998; Pasqualucci et al., 2001; Muschen et al., 2000; Gordon et al., 2003). The significance of these mutations is not known. RhoH, however, is not hypermutated in normal germinal B cells. Interestingly, the tumour specific mutations of the RhoH gene are introduced into the 5’ untranslated region, in the intron between exon 1b and exon 2. This further suggests that RhoH expression might be deregulated in lymphomas (Pasqualucci et al., 2001).
The expression analysis of the human RhoH gene in cell lines of different hematopoietic lineages demonstrated transcriptional regulation of the RhoH gene (Lahousse et al., 2004). The human RhoH gene consists of 7 exons (X1, 1a, X2, 1b, X3, X4, 2) and the coding region is located within exon 2 (Figure 14; Lahousse et al., 2004). The gene structure is characterized
by an intronless open reading frame (ORF), preceded by a very large 5’ region about 50 kb in length. Several transcription initiation sites were detected, and a high 5’ end heterogeneity of mRNAs was observed due to alternative splicing of some 5’ exons. Moreover, different transcription start sites are used in different hematopoietic cell lines (Figure 14; Lahousse et al., 2004; Preudhomme et al., 2000). In addition, RhoH might be controlled at the post- transcriptional level since multiple polyadenylation sites and “AU”-rich elements were identified, which could regulate mRNA stability by differential polyadenylation. The presence of two transcripts at 1.8 and 2.2 kb indeed might reflect the usage of two different polyadenylation sites.
Figure 14. The RhoH gene structure and mRNA isoforms in different hematopoietic cell lines. Thehuman RhoH gene consists of
7 exons. The ORF is located in exon 2. Sizes of exons and introns are indicated (C). (A) Patterns of major RhoH mRNAs isoforms. Two major isoforms A (“1b-X4-2”) and C (“1a-X4-2”) are present in cell lines from all hematopoietic lineages. B (“1a-1b-X4-2) is only present in B lymphoid and erythroid cell lines. (B) Patterns of minor RhoH mRNAs isoforms. All seven minor species (d-j) were detected in the B cell line Raji. E: erythroid, M: myeloid, LT: T lymphoid, B: B lymphoid (modified from Lahousse et al., 2004).
The transcriptional regulation of RhoH was demonstrated in Jurkat cells, a human T cell leukemia line, and in T cells. In Jurkat cells, phorbol-12-myristate-13-acetate (PMA) treatment decreased RhoH mRNA by 80% within 60 to 80 min, and the activation of the TCR in T cells reduced RhoH transcript levels within a few hours (Li et al., 2002).
At highly conserved positions in the aa sequence RhoH has different aa when compared to other Rho GTPases: at the position 13, serine instead of glycine and at the position 62, asparagine instead of glutamine. The aa glycine and glutamine are crucial for GTP hydrolysis (Figure 15). Similar to RhoH, Rnd3/RhoE GTPase has also different aa at these positions (serines at both positions) and is known to be deficient in GTPase activity. Therefore, it is assumed that RhoH has no intrinsic GTPase activity and is constitutively active. RhoH was
shown that it cannot autohydrolyze GTP and cannot hydrolyze GTP in the presence of RhoGAP p50, a potent GAP for RhoA, Rac and Cdc42 (Li et al., 2002).
RhoH was demonstrated to bind to all three known GDIs, which are capable of extracting Rho GTPases from membranes, implying a possible role in regulation of subcellular localization of RhoH. At the C-terminus, RhoH contains a typical CAAX motif (SKIF), which is most likely geranylated, like other Rho GTPases. Such geranylation should allow its association with membranes. However, RhoH was found to be mainly distributed to the cytoplasm (Li et al., 2002).
Functionally, RhoH was proposed to be a negative regulator of Rho GTPases. RhoH anta- gonizes Rac1-, RhoA- and Cdc42-mediated activation of NF-κB and p38 when transfected into Jurkat and 293 cells, while having no effect on JNK or Erk in these cells (Li et al., 2002). In addition, RhoH overexpression in HPCs suppresses Rac1 activation in response to stem cell factor (Gu et al., 2005a).
In contrast to other Rho GTPases, RhoH does not exert a significant role on the actin cytoskeleton reorganization in non-hematopoietic 293 cells (Li et al., 2002). However, in HPCs, overexpression of RhoH is associated with defective assembly and polarization of F- actin and reduced chemokine-induced cell migration in vitro (Gu et al., 2005a). Furthermore, in hematopoietic Jurkat cells and human peripheral blood cells, RhoH was demonstrated to be required to maintain the integrin lymphocyte function-associated antigen 1 (LFA-1), αLβ2- integrin, in a non-adhesive state (Cherry et al., 2004).
Retrovirus-mediated overexpression of RhoH resulted in reduced activity of HPCs to engraft into the BM of lethally irradiated mice, as well as in decreased HPC proliferation and increased HPC apoptosis in vivo. Knockdown of RhoH expression in HPCs led to increased HPCs proliferation, survival and migration activities, implying that RhoH is a negative regulator of proliferation and survival (Gu et al., 2005a).
PAK5, a member of the serine-threonine protein kinase family, has recently been identified as an effector protein binding to RhoH (Wu and Frost, 2006). PAK5 localizes to mitochondria and is thought to be important for protecting cells from apoptosis (Cotteret et al., 2003; 2006). However, the biological function of the interaction between RhoH and PAK5 is unclear (Wu and Frost, 2006).
In conclusion, the Rho GTPase family member RhoH displays a hematopoietic expression, preferentially in lymphoid cells and to a lesser extent in myeloid lineages (Li et al., 2002; Gu et al., 2005a; Lahousse et al., 2004). RhoH was suggested to be constitutively active and thus regulated transcriptionally (Li et al., 2002). Deregulation of RhoH expression probably occurs
in various lymphomas, where the RhoH gene is often mutated in the non-coding region or translocated into the locus of other genes (Pasqualucci et al., 2001; Preudhomme et al., 2000). Functionally, RhoH was shown to inhibit Rho GTPases and integrins (Li et al., 2002; Gu et al., 2005a; Cherry et al., 2004).