Paul R. Kemp

Active and acid-activatable TGF-β in human sera, platelets and plasma

Assays which measure active and latent forms of transforming growth factor beta (TGF-beta) separately in human serum and plasma are required to investigate the biological role of TGF-beta in a variety of human diseases. We have developed an enzyme-linked immunosorbent assay (ELISA) using two polyclonal antibodies against TGF-beta which rapidly determines the amount of active plus acid-activatable, latent TGF-beta forms ((a+l)TGF-beta) present in human serum and plasma in the range 4 pmol/l to 2000 pmol/l. To measure active TGF-beta alone, we have developed a second ELISA using the extracellular domain of the TGF-beta type II receptor as the capture reagent which detects active TGF-beta in serum and plasma samples in the range 20 pmol/l to 4000 pmol/l. Both assays detect TGF-beta 1 and TGF-beta 3 with similar sensitivity, are > 10-fold less sensitive to TGF-beta 2 and are not affected by a range of other peptide growth factors. The mean (a+l)TGF-beta present in human serum was 330 pmol/l but the range was very large (< 4 pmol/l to 1400 pmol/l). The mean active TGF-beta present was 230 pmol/l (range < 20 pmol/l to 1400 pmol/l) and the proportion of the (a+l)TGF-beta present which was active [a/(a+l)] varied from < 10% to 100%. The concentration of (a+l)TGF-beta and the proportion of TGF-beta which was active were very similar in the serum and platelet-poor plasma prepared from the same whole blood sample. The clot formed during serum preparation retained all of the TGF-beta which was detected by the (a+l)TGF-beta ELISA in the corresponding platelet releasate, although the PDGF in platelets was released into the serum. In contrast, platelet-poor plasma contained no detectable PDGF demonstrating that the (a+l)TGF-beta assayed in the plasma was not due to platelet degranulation after bleeding. Serum active TGF-beta and (a+l)TGF-beta concentrations therefore provide a reliable estimate of these forms of TGF-beta present in plasma.

Effects of premature stimulation on HERG K+ channels

1 The unusual kinetics of human ether‐à‐go‐go‐related gene (HERG) K+ channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K+ channels, transfected in Chinese hamster ovary cells, at 37 °C. 2 HERG K+ channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). 3 HERG K+ channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90% repolarization (APD90) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD90– 20 ms) and atrial (APD90– 30 ms) action potentials. 4 The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. 5 A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre‐open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. 6 These properties of HERG K+ channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria.

Cytoplasmic YY1 Is Associated with Increased Smooth Muscle-Specific Gene Expression Implications for Neonatal Pulmonary Hypertension

Immediately after birth the adluminal vascular SMCs of the pulmonary elastic arteries undergo transient actin cytoskeletal remodeling as well as cellular de-differentiation and proliferation. Vascular smooth muscle phenotype is regulated by serum response factor, which is itself regulated in part by the negative regulator YY1. We therefore studied the subcellular localization of YY1 in arteries of normal newborn piglets and piglets affected by neonatal pulmonary hypertension. We found that YY1 localization changed during development and that expression of gamma-smooth muscle actin correlated with expression of cytoplasmic rather than nuclear YY1. Analysis of the regulation of YY1 localization in vitro demonstrated that polymerized gamma-actin sequestered EGFP-YY1 in the cytoplasm and that YY1 activation of c-myc promoter activity was inhibited by LIM kinase, which increases actin polymerization. Consistent with these data siRNA-mediated down-regulation of YY1 in C2C12 cells increased SM22-alpha expression and inhibited cell proliferation. Thus, actin polymerization controls subcellular YY1 localization, which contributes to vascular SMC proliferation and differentiation in normal pulmonary artery development. In the absence of actin depolymerization, YY1 does not relocate to the nucleus, and this lack of relocation may contribute to the pathobiology of pulmonary hypertension.

Loss of a consensus heparin binding site by alternative splicing of latent transforming growth factor-β binding protein-1

Latent transforming growth factor‐β binding protein‐1 (LTBP‐1), plays an important role in controlling localisation and activation of transforming growth factor‐β (TGF‐β). We show that alternative splicing generates a form of mRNA which lacks bases 1277–1435 (termed LTBP‐1Δ53). The 53 amino acids encoded by these bases include the eighth cysteine of the first cysteine repeat and a consensus heparin binding sequence. Sequencing of genomic clones showed that alternative splicing resulted from the use of an intra‐exonic 3′ splice acceptor site. The loss of the heparin binding site implies that LTBP‐1Δ53 will bind to the extracellular matrix less efficiently than LTBP‐1.

Mouse BTEB3, a new member of the basic transcription element binding protein (BTEB) family, activates expression from GC-rich minimal promoter regions.

Members of the three-zinc-finger family of transcription factors play an important role in determining basal transcription. We have cloned mouse BTEB3 (mBTEB3), a new member of the basic transcription element binding protein (BTEB) family, which is expressed in a wide variety of tissues. mBTEB3 activates transcription of the simian virus 40 early promoter (4-fold) and of the tissue-specific SM22alpha promoter (100-fold), suggesting that, like BTEB1 and Sp1, mBTEB3 is a basal transcription factor.

Increased actin polymerization reduces the inhibition of serum response factor activity by Yin Yang 1.

Recent evidence has implicated CC(A/T(richG))GG (CArG) boxes, binding sites for serum response factor (SRF), in the regulation of expression of a number of genes in response to changes in the actin cytoskeleton. In many cases, the activity of SRF at CArG boxes is modulated by transcription factors binding to overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites. However, the mechanisms by which SRF activity is regulated by the cytoskeleton have not been determined. To investigate these mechanisms, we screened for cells that did or did not increase the activity of a fragment of the promoter for a smooth-muscle (SM)-specific gene SM22alpha, in response to changes in actin cytoskeletal polymerization induced by LIM kinase. These experiments showed that vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters containing at least one of the SM22alpha CArG boxes (CArG near) in response to LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in contrast with the other two cell types. Expression of YY1 in P19 cells inhibited SM22alpha promoter activity and conferred responsiveness to LIM kinase. Mutation of the CArG box to inhibit YY1 or SRF binding indicated that both factors were required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate that changes in the actin cytoskeletal organization modify SRF activity at CArG boxes by modulating YY1-dependent inhibition.

Adhesion of endothelial cells to NOV is mediated by the integrins αvβ3 and α5β1

NOV is a member of the CCN family of matricellular proteins. We have shown previously that Nov is strongly expressed by vascular smooth muscle cells (VSMCs) of the rat carotid artery. However, 7 days after injury, Nov expression is down-regulated, except near the luminal surface of the developing intima, where it is strongly expressed. These data suggested that NOV might be involved in the regulation of endothelial cell adhesion. NOV promoted the adhesion of human umbilical vein endothelial cells (HUVECs), which was abolished by anti-NOV antibody. HUVEC adhesion to NOV required divalent cations and was inhibited by GRGDS peptide, implicating integrins in the adhesion mechanism. Monoclonal antibodies (mAbs) against αvβ3 inhibited adhesion of HUVECs to NOV, and NOV was shown to bind to αvβ3. Anti-α5β1 mAbs also inhibited HUVEC adhesion to NOV, but adhesion via α5β1 was mediated by fibronectin. HUVEC adhesion to NOV caused intracellular signalling, as evidenced by increased phosphotyrosine content of focal adhesion kinase. Together with evidence that Nov expression in a variety of tissues is restricted to blood vessels containing VSMCs, these data are consistent with a role for NOV in endothelial cell adhesion in vascular homeostasis and in the response to injury.

Splenomegaly and Modified Erythropoiesis in KLF13–/– Mice

To study the function of the Krüppel-like transcription factor KLF13 in vivo, we generated mice with a disrupted Klf13 allele. Although Klf13–/– mice are viable, fewer mice were present at 3 weeks than predicted by Mendelian inheritance. Viable Klf13–/– mice had reduced numbers of circulating erythrocytes and a larger spleen. The spleen contained an increased number of Ter119medCD71hi, Ter119hiCD71hi, and Ter119hiCD71med cells but not Ter119hiCD71– cells, indicating an increase in less mature erythroblasts. A higher proportion of the Ter119medCD71hi cells were proliferating, indicating that the mice were under a degree of erythropoietic stress. These data indicate that KLF13 is involved in the normal control of erythropoiesis.

Expression of alternatively spliced human latent transforming growth factor β binding protein-1

Latent transforming growth factor β binding protein‐1 (LTBP1) is important in regulating the localisation and activation of transforming growth factor β (TGFβ). Three forms of LTBP1 mRNA have previously been described, LTBP1L, LTBP1S and LTBPΔ53. Here, we have analysed the LTBP1 coding sequence and identified two other spliced forms, LTBP1Δ55 and LTBP1Δ41. LTBP1Δ55 is a short form of LTBP1L which lacks 55 amino acids including two consensus N‐glycosylation sites and LTBP1Δ41 is a form of LTBP1 which lacks the 12th EGF‐like repeat. Furthermore, sequencing of genomic clones showed that splicing to generate LTBP1L occurs using an intra‐exonic 3′ splice acceptor site in the first coding exon of LTBP1S and that LTBP1Δ55 arises from the alternative use of an exonic 3′ splice acceptor site at the end of the following intron. LTBP1Δ41 arises from skipping the exon which encodes the 12th EGF‐like repeat. LTBP1Δ55 and LTBP1Δ41 mRNA are expressed in a wide variety of human tissues but the proportions of each splice form vary in the tissues.

KLF13 influences multiple stages of both B and T cell development

The Kruppel-like factor, KLF13, is a member of a family of transcription factors shown to be involved in haematopoietic development. Here we show that KLF13 is involved in the development of B and T cells at multiple stages. Expression of KLF13 in the thymus was maximal in the DP population and in KLF13-/- deficient mice there was an accumulation of DP thymocytes and reduction of CD4+SP cells. Cell-surface expression of CD3high, CD8, CD5 and HSA were altered on KLF13-/- DP cells, consistent with a defect in TCR signalling and at the DP to SP transition in KLF13-/- mice. KLF13 is also expressed in peripheral T-cells and peripheral T cell activation was impaired in KLF13-/- mice. Analysis of early B cell development in the bone marrow (BM) revealed a partial arrest of B cells at the transition from CD43+ to CD43- pre-B cell, a transition that requires signalling through the pre-BCR. The proportion of IgM+/IgD+ mature B cells was also increased in the BM of the KLF13-/- mice. This finding is consistent with a reduction in the strength of BCR signal or an accumulation of recirculating B cells from the periphery. Analysis of splenocytes isolated from KLF13-/- mice revealed an increase in the expression of CD21 and CD23 on B220+ B cells, demonstrating a negative regulatory role for KLF13 in co-regulation of expression of CD21 and CD23. Thus KLF13 is involved at multiple different checkpoints in development that require signalling through the TCR, pre-BCR or mature BCR.