HC0₃⁻ plays a vital role in the airways, as mucus viscosity and ciliary beat have both been shown to be dependent upon the pH of the airway surface liquid. In cystic fibrosis (CF), reduced HC0₃⁻ and fluid secretion produce dehydrated mucus, which impairs airway defence and predisposes the airways to bacterial infection. Calu-3 cells are used as a model of the serous cells of human tracheobronchial submucosal glands which are involved in CFTR (cystic fibrosis transmembrane conductance regulator)dependent HC03⁻ secretion. CFTR is widely regarded as the sole mediator of apical C1⁻ and HC0₃⁻ secretion in Calu-3 cells and human serous airway cells. However, the discovery ofSLC26 C1⁻/HC0₃⁻ exchange activity linked to the expression of CFTR in tracheal epithelial cells, as well as a functional interaction between apically co-localised SLC26 exchangers and CFTR in mediating HC03- secretion in many HC0₃⁻ secreting epithelia including the pancreas and gastrointestinal tract, has led to some doubt over this hypothesis. The aim of this work was to investigate the potential role of SLC26 anion exchange (AE) in HC0₃⁻ secretion from Calu-3 cells and its dependence on CFTR. AE activity was assessed by real time measurements of intracellular pH (pHi) using the pH-sensitive dye BCECF-AM, from cells grown as monolayers on semi-permeable supports. Calu-3 cells under non-stimulated conditions displayed Na⁺-dependent basolateral C1⁻/HC0₃⁻ exchange, which was abolished by H₂DIDS. No apical AE activity could be detected under resting conditions. Stimulation of cells with a cAMP agonist (forskolin, adenosine, VIP) produced a switch in AE activity, activating an apical AE and completely inhibiting the basolateral AE. The cAMP-activated apical AE activity was Na⁺-independent, H₂-DIDS-insensitive and could transport a range of monovalent anions in exchange for HC0₃⁻ with the selectivity profile: Iodide=Br -> Cr=Formate =N0₃⁻=SCN-. The profile of this anion exchanger corresponds with Pendrin (SLC26A4), which quantitative RT-PCR analysis showed to be expressed in these Calu-3 cells. Consistent with this, Pendrin knockdown (KD) Calu-3 cells had a reduced rate of AE activity, compared to wild-type (WT) cells. Protein phosphatase 1 inhibition by okadaic acid activated an apical AE under non-stimulated conditions with a similar profile to Pendrin, which CFTR inhibitor studies demonstrated to be CPTR-independent. Interestingly, fluid secretion studies established that Calu-3 cells produced a more alkali fluid when treated with okadaic acid, consistent with enhanced HC0₃⁻ secretion. The role of CFTR in C1⁻HC0₃⁻ AE activity was assessed using CFTR inhibitors and CFTR KD Calu-3 cells. Although apical AE activity in WT Calu-3 cells could be abolished by inhibiting CFTR (with either CFTRinh-172 or GlyH-lOl), it could be restored by the addition of basolateral H₂-DIDS, suggesting that the CFTR inhibition reveals a H₂-DIDS-sensitive basolateral transport process which masks the effects of apical AE on pHi, rather than abolishing apical AE activity itself. In CFTR KD Calu-3 cells the rate of apical AE activity was significantly decreased compared to WT cells. These results suggest a role for CFTR in regulating apical C1⁻HC0₃⁻ exchange activity and/or contributing to C1⁻dependent HCO₃⁻ transport. Similarly cAMP inhibition of the basolateral AE was also found to be mediated via a CFTR-dependent mechanism, highlighting the importance of CFTR in the cAMP-dependent switch in Calu-3 AE activity. Such a switch in AE activity by cAMP, would favour bicarbonate secretion, consistent with the finding that cAMP enhances HC0₃⁻-dependent fluid secretion from Calu-3 cells. Apical AE activity was also observed in human bronchial epithelial cells (HBECs), which could not be blocked by GlyH-101, but was absent in CF HBECs.

Tubulo-interstitial fibrosis (TIF) is a key feature of chronic kidney diseases (CKD). Epithelial mesenchymal transition of PTECs is considered to contribute to the interstitial fibroblast pool which secretes excessive matrix proteins seen in TIF. TGFpl plays a crucial role in TIF including mediating EMT. In animal models of CKD treatment with bone morphogenic protein 7 (BMP 7), a member of TGF superfamily improved the histology and renal function. However, the cellular signalling mechanisms involved in the anti- fibrotic effects of BMP 7, in particular whether BMP 7 inhibits TGFp 1 mediated EMT of human PTECs have not been studied. Experiments were performed in HKC 8 cells; a virally transformed human PTEC model. The expression of cell surface receptors for TGF superfamily and phosphorylation of TGFp 1 and BMP 7 Smads were studied by immunoblotting, and the nuclear translocation of Smad proteins was studied by immunofluorescence. The expressions of E-cadherin and (l-SMA were studied with TGFp 1 and BMP 7 treatment individually and in combination. siRNAs were used to knock-down Smadl and 5 proteins, to identify their role in BMP 7 regulation of markers of EMT. TGFp 1 and BMP 7 regulation of Id2 expression was studied at the protein level and the Smad signalling regulating this process was studied by '. silencing their expression with siRNA. siRNAs were used to study the role of Id2 on the expression of E-cadherin and (l-SMA with TGFp 1 and BMP 7 stimulation. Plasmid vector expressing Id2 was used to overexpress Id2 to study its role in TGFp 1 regulation of , markers ofEMT. Idl expression was studied at the protein level with BMP 7, and the Smad signalling involved in this process was studied by silencing their expression with siRNAs. The role of Id 1 in BMP 7 regulation of E-cadherin and (l-SMA was studied by silencing its expression by siRNA. The interaction of Idl and Id2 with E2A gene products was studied by nuclear co-immunoprecipitation. HKC 8 cells express TGF type 11, Alkl, Alk2, Alk3, Alk5 and Alk6 receptors. TGFpl and BMP 7 activated their respective receptor Smads concurrently during combined treatment. The nuclear translocation of their respective receptor Smads was not inhibited by the other during combined treatment. BMP 7 do~egulated E-cadherin, and it had an additive effect with TGFp 1 in this process and this was a Smad 1/5 dependent event. BMP 7 inhibited TGFp 1 induction of (l-SMA through Smad 1/5 signalling. TGFp 1 downregulated Id2 through Smad2/3 signalling and BMP 7 counter-regulated this through Smad1/5 signalling. IV Id2 gene silencing prevented BMP 7 inhibition of TGF~ I mediated a-SMA' expression. Id2 overexpression prevented TGF~ I mediated a-SMA expression. BMP 7 increased expression of Idl through non-Smadl/5 pathway and Id l gene silencing resulted in induction of a-SMA with BMP 7 stimulation. Both Idl and Id2 were co- immunoprecipitated with EI2 and E47 in the nuclear extract. In conclusion, these studies show that the anti-fibrotic effect of BMP 7 is mediated by inhibition of TGF~ 1 mediated induction of a-SMA and the myofibroblastic transition of PTECs in this in vitro model. The activation of Smadl/5 and Id2 signalling is involved in this counter-regulation. Although BMP 7 itself downregulated E-cadherin through Smadl/5 signalling, the concurrent induction of Idl through non-Smadl/5 pathway prevented de novo induction of a-SMA and myofibroblastic transition of PTECs. Id I and Id2 were shown to bind with E2A gene products, but their role in the expressions of markers of EMT in this model needs further confirmation.

Transglutaminase type 2 (TG2) catalyses the formation of an ε-(γ-glutamyl)-lysine isopeptide bonds between adjacent peptides or proteins including those of the extracellular matrix(ECM). ECM crosslinking has been associated with both the acceleration of collagen deposition while conferring the ECM with resistance to proteolytic degradation. Subsequently the cellular secretion of TG2 has been associated with wound healing and aberrant wound healing leading to kidney, lung, liver and heart fibrosis as well as atherosclerosis. TG2 has no signal peptide and cannot be transported classically. It is unknown how TG2 is targeted to the cell surface and secreted into ECM. Understanding TG2 transport may help to develop specific mechanisms to interfere with TG2 action in the scarring process. In this study, we identified that amino acids 88-106 in N-terminal β-sandwich domain of TG2 molecule is crucial for TG2 externalisation using deletion and mutation analysis in three renal tubular epithelial cells (TEC). Of interest, this TG2 export motif (aa88-106) itself appeared to be able to target other proteins for extracellular secretion. Yeast-two-hybrid studies were then performed to identify what the TG2 export motif would bind to and thus give clues as to the downstream mechanism of trafficking. Large T antigen (LTA) and tapasin were identified as binding partners. The interaction between LTA or tapasin and TG2 was confirmed by co-immunoprecipitation using endogenous protein from wild-type cells. TG2 externalisation was significantly decreased when LTA and tapasin were knockdown using siRNA suggesting that large T antigen and tapasin is involved in TG2 externalisation process. The possible TG2 externalisation pathway was explored further using fluorescent imaging including co-localisation analysis and live cell imaging. TG2 was predominantly co-localised with endoplasmic reticulum (ER) around the cell nucleus, but not localised with Golgi apparatus and lysosomes. We observed plasma membrane blebbing in the cells transfected with wild-type TG2 but not in the cells transfected withTG2 carrying a mutation in the export motif. Plasma membrane blebbing or a direct molecular trap is the most likely mechanism for TG2 externalisation based on the data generated. In conclusion, the amino acid sequence 88-106 in β-sandwich domain of TG2 is critical to TG2 externalisation in TEC. This export motif binds to large T antigen and tapasin. Large T antigen and tapasin is involved in TG2 externalisation possibly through plasma membrane blebbing or direct molecular trap in TEC.

Background: RNA splicing is a crucial process for delivering the appropriate message for protein synthesis. Most genes are affected by alternative splicing, and among these is CHRNA7. This gene encodes for the nicotinic acetylcholine α7 receptor subunit that is involved in the cholinergic anti-inflammatory pathway. This anti-inflammatory pathway is considered an important part of the human body’s defence line against tissue injury or infection and causative mechanism in COPD. Aim: The aim of the present study was to investigate the role of alternative splicing on the nature of the transcripts generated by CHRNA7 gene and its partial duplicate, CHRFAM7A. Methods: Airway epithelial cell lines, A549 and BEAS2B, were mainly used as targets for testing alternative splicing. RT-PCR, TA cloning and gel extraction methods were used for testing CHRNA7 and CHRFAM7A transcripts. Following RT-PCR, the resulting product band intensities were analysed using densitometric analysis tools. This was followed by the use of several bioinformatics analysis tools to predict the protein structure for the resulting transcripts. For one of the detected transcripts, minigene methods were used to test for the source of expression. Results: A novel transcript missing exon 9 is reported for the first time. Both genes showed the expression of full length and the novel transcripts (missing exon 9) at similar ratios (~2:1). These results could be detected in immortalised cell lines from human alveolar and bronchial epithelial cells (A549 and BEAS2B, respectively) and in BE (2)-c cells (neuroblastoma cells with bone marrow metastasis). The same results were shown when primary human peripheral blood monocytes cells (PBMC) were tested. This means that the effect of missing exon 9 is not tissue-specific, and is not only found in cancerous cells, indicating that it could be a common feature of splicing for these two genes. Furthermore, another novel transcript was detected which is inserted exon 9b. The initial RT-PCR experiments seemed to suggest that this was derived from CHRFAM7A only. The use of minigene methods showed that this transcript could be expressed from both genes, CHRNA7 and CHRFAM7A, but a single nucleotide base within the inserted sequence (at position 77 from the 5` end) could play a role in enhancing of exon 9b in the mRNA transcripts. This base is C allele in CHRFAM7A sequence of exon 9b, while its corresponding base in CHRNA7 is G allele that has less prominent effect on exon 9b inclusion. Conclusion: CHRNA7 and CHRFAM7A express novel transcripts in different human cells that are missing exon 9. This could be due to inactive splicing factors that are required for recognition of exon 9 as a constitutive exon. For exon 9b transcripts, these lie within the common sequence of CHRNA7 and CHRFAM7A, and it seems that the presence of C allele at position 77 could enhance the inclusion of exon 9b in CHRFAM7A more than the presence of G allele in CHRNA7 sequences. The results shown in this study implicate a possible regulatory role of the transcripts detected on the control mechanism exerted by CHRFAM7A on CHRNA7. These results help to suggest a possible role of in the development of COPD in the form of inflammatory/anti-inflammatory control imbalance.