Sreedharan Kartha

Sequence of a human kidney cDNA clone encoding thymosin β10

Summary We have isolated a cDNA clone encoding the 43 amino acid protein, thymosin β 10 , from an adult human kidney cDNA library. A comparison of human and rat thymosin β 10 cDNA sequences revealed 100% identity for the deduced amino acid sequence and 95% nucleotide identity for the coding region. The thymosin β 10 cDNA clone was used to probe RNA isolated from monkey kidney epithelial cells of the BSC-1 line. The probe recognized 850 bp and 2.4 kb transcripts in quiescent cells which indicates that the gene is constitutively expressed. The abundance of the 2.4 kb transcript was markedly increased one hour after serum stimulation and declined progressively to a basal value at 24 hours.

AMP-18 facilitates assembly and stabilization of tight junctions to protect the colonic mucosal barrier.

Background: Inflammatory bowel disease (IBD) is characterized by an injured epithelium. Development of agents that could enhance mucosal healing is a major goal in IBD therapeutics. The 18‐kDa antrum mucosal protein (AMP‐18) and a 21‐mer peptide derived from AMP‐18 stimulate accumulation of tight junction (TJ) proteins in cultured epithelial cells and mouse colonic mucosa to protect the mucosal barrier, suggesting it might be a useful agent to treat IBD. Methods: We searched for molecular mechanisms by which AMP peptide or recombinant AMP‐18 act on TJs in intact cell monolayers, or those disrupted by low‐calcium medium. Roles of the p38 mitogen‐activated protein kinase (MAPK) / heat shock protein (hsp)25 pathway and PKC&zgr; were investigated by immunoblotting and confocal microscopy. Results: AMP peptide activated p38 MAPK, which subsequently phosphorylated hsp25. Accumulated phospho‐hsp25 was associated with perijunctional actin. AMP‐18 also induced rapid phosphorylation of PKC&zgr; and its colocalization with perijunctional actin in Caco2/bbe cells, which was accompanied by translocation and formation of complexes of “polarity proteins” in the TJ‐containing detergent‐insoluble fraction. Treatment with AMP‐18 also stimulated accumulation of ZO‐1, ZO‐2, and JAM‐A in nascent TJs known to associate with the multimeric p‐PKC&zgr;/Par6/ Cdc42/ECT2·GTP/Par3 polarity protein complex. Conclusions: AMP‐18 facilitates translocation and assembly of multiple proteins into TJs and their association with and subsequent stabilization of the actin filament network. We speculate that improved barrier function induced by AMP‐18 is mediated by enhanced TJ assembly. Thus, AMP‐18 may provide a promising lead to develop agents effective in healing injured colonic epithelium in IBD. (Inflamm Bowel Dis 2012;)

Kidney epithelial cells release growth factors in response to extracellular signals

The growth of nontransformed monkey kidney epithelial cells in culture appears to be regulated by the interplay of positive and negative autocrine growth factors. Reduction of the potassium or sodium concentration of the medium induces rapid release of novel growth-promoting activities, whereas addition of the mitogen adenosine diphosphate stimulates the appearance of a platelet-derived growth factor-like protein which could function in a paracrine manner. These observations suggest that autocrine and paracrine growth factors could play an important role in physiological and pathological states in the kidney.

Cytokeratin reorganization induced by adenosine diphosphate in kidney epithelial cells

Exogenous adenosine diphosphate (ADP), the most potent mitogen for nontransformed African green monkey kidney epithelial cells of the BSC-1 line, rapidly alters the appearance of the cell monolayer. Examination of the cells with indirect immunofluorescence using monoclonal antibodies reveals a considerable reorganization of cytokeratin filaments without a major change in the pattern of microtubules or microfilaments. In untreated confluent cells, cytokeratin filaments are predominantly confined to a star-like spot in the perinuclear area, but these can be seen to begin to spread within 2 min after addition of ADP. The effect is particularly notable using anti-cytokeratin 8 antibodies. At 6 h this process is complete and produces a well-developed filamentous network throughout the cell. By 12 h, the network appears to collapse, so that the filaments again form a spot in the perinuclear area, a process that is complete by 24 h. Immunoblotting of total cellular proteins reveals no apparent alterations in the amounts of several species of cytokeratins, including cytokeratin 8 and 18, at 3 or 24 h after exposure to ADP. Other purine and pyrimidine nucleotides which do not stimulate DNA synthesis in these cells fail to alter cytokeratin organization, and there is no apparent alteration in the distribution of vimentin, another intermediate filament protein. The rapid ADP-induced cytokeratin reorganization appears to coincide with the induction of early growth-response gene transcription in these cells and may be correlated with the capacity of ADP to subsequently initiate DNA synthesis. This dramatic and reversible cytokeratin reorganization immediately after exposure to ADP may be an important step in the mitogenic signal transduction pathway.

Regulation of Delta-Aminolevulinic Acid Dehydratase by Krüppel-Like Factor 1

Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the β-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.

Signals That Release Growth Factors From Renal Epithelial Cells

Monkey kidney epithelial cells of the nontransformed BSC-1 line have been used as a model system to investigate growth control. Renal growth in K depletion nephropathy was studied in culture by reducing the K concentration of the medium, which accelerated cell proliferation. This response appeared to be mediated by release of a growth-promoting activity that has an apparent molecular weight of 12,000 to 30,000. Growth stimulation was also observed when the Na concentration of the medium was reduced and was associated with the appearance of two growth-promoting factors (apparent molecular weight, 6,200 and 9,000) that exhibited cell-type specificity. Thus, modest reductions in the extracellular concentration of K or Na result in rapid appearance of autocrine factors that could modulate cell function along the nephron. The most powerful mitogen for BSC-1 cells is adenosine diphosphate (ADP). This nucleotide stimulates expression of several cell cycle-specific genes and proto-oncogenes, and induces secretion of a platelet-derived growth factor-like protein that is not mitogenic for BSC-1 cells. Release of this growth factor by renal epithelial cells in vivo would represent a paracrine mechanism to initiate proliferation of neighboring stromal or vascular cells.

Determinants of Autocrine and Paracrine Growth Factor Release by Kidney Epithelial Cells

During studies of mitogenic signal transduction mechanisms in nontransformed monkey kidney epithelial cells in culture, evidence for the release of auto-crine and paracrine growth factors was obtained. Reduction of the K or Na concentration of the culture medium induced rapid release of novel growth-promoting activities, whereas addition of the mitogen, adenosine diphosphate, stimulated the appearance of a platelet-derived growth factor-like protein which could function in a paracrine manner. These cells constitutively release transforming growth factor β2, which acts as a negative autocrine growth factor. Thus, growth in culture appears to be regulated by the interplay of positive and negative autocrine growth factors and the responses of cell surface receptors and post-receptor events. Autocrine and paracrine growth factors released in response to alterations in the ionic and nutrient composition of the extracellular environment could play an important role in physiological and pathological states in the kidney.