Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. AUl of the amino acid substitutions were clustered in the amino-terminal domain ofthe protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity.Protein phosphorylation is a major regulatory mechanism for the control of cellular metabolism, growth, and differentiation (1-3). A number of protein kinases have been identified that specifically phosphorylate various protein substrates. Phosphoserine and phosphothreonine protein phosphatases have been broadly categorized as either type 1 or type 2 based on their sensitivity to inhibitors and substrate specificity (4). The type 2 phosphatases have been further divided into three groups termed 2A, 2B, and 2C (5). Little structural information has been available for the catalytic subunits of the protein phosphatases, but protein sequencing (6) and isolation of appropriate cDNAs (7-9) have indicated there are significant homologies between the type 1 and type 2 protein phosphatases, suggesting they have been highly conserved in primary sequence.By using a partial cDNA probe to bovine phosphatase 2A (7) Tris HCI (pH 8.0), 0.9 M NaCi, 6 mM EDTA, 5 x Denhardt's solution (0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% bovine serum albumin), 10% (wt/vol) dextran sulfate, 0.1% NaDodSO4, and the radiolabeled probe (6 x i0' cpm/ml).The nitrocellulose filters were washed twice in 2 x SSC at room temperature for 10 min and once at 530C for 30 min.(1 x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0.)Recombinant phage giving positive signals were plaquepurified. DNA prepared from positive recombinant phage was isolated and the inserts were subcloned into pUC18 and M13mpl8 vectors. At this point, restriction analysis of the clones was carried out by incubation with the appropriate restriction endonucleases followed by electrophoretic analysis in agarose gels (12).The nucleotide sequence of the clones was established after subcloning the appropriate restriction fragments into M13mpl8 or M13mpl9 phage (13). All sequencing reactions were performed by the dideoxy chain-termination method (14) with modified T7 DNA polymerase (15), and both strands were sequenced in all cases. The sequencing of the HL-1 and HL-14 clones was also performed with the Cyclone system (International Biotechnologies, New Haven, CT) to progressively del...
Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel-like factor 15 (KLF15), a kidney-enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15 In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone-induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.
Background/Aims: Short stature is a common reason for presentation to pediatric endocrinology clinics. However, for most patients, no cause for the short stature can be identified. As genetics plays a strong role in height, we sought to identify known and novel genetic causes of short stature. Methods: We recruited 14 children with severe short stature of unknown etiology. We conducted whole exome sequencing of the patients and their family members. We used an analysis pipeline to identify rare non-synonymous genetic variants that cause the short stature. Results: We identified a genetic cause of short stature in 5 of the 14 patients. This included cases of floating-harbor syndrome, Kenny-Caffey syndrome, the progeroid form of Ehlers-Danlos syndrome, as well as 2 cases of the 3-M syndrome. For the remaining patients, we have generated lists of candidate variants. Conclusions: Whole exome sequencing can help identify genetic causes of short stature in the context of defined genetic syndromes, but may be less effective in identifying novel genetic causes of short stature in individual families. Utilized in the clinic, whole exome sequencing can provide clinically relevant diagnoses for these patients. Rare syndromic causes of short stature may be underrecognized and underdiagnosed in pediatric endocrinology clinics.
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