Limited phenotypic variability has been re- ported in patients with Bartter syndrome type I, with mu- tations in the Na-K-2Cl cotransporter gene ( BSC). The diagnosis of this hereditary renal tubular disorder is usu- ally made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, hypo- kalemia, metabolic alkalosis, hypercalciuria, and nephro- calcinosis. Among nine children with hypercalciuria and nephrocalcinosis, we identified new mutations consistent with a loss of function of the mutant allele of the BSC gene in five. Three of the five cases with BSC gene mu- tations were unusual due to the absence of hypokalemia and metabolic alkalosis in the first years of life. The di- agnosis of incomplete distal renal tubular acidosis was considered before molecular evaluation. Three additional patients with hypokalemia and hypercalciuria, but with- out nephrocalcinosis in the first two and with metabolic acidosis instead of alkalosis in the third, were studied. Two demonstrated the same missense mutation A555T in the BSC gene as one patient of the previous group, sug- gesting a single common ancestor. The third patient pre- sented with severe hypernatremia and hyperchloremia for about 2 months, and a diagnosis of nephrogenic dia- betes insipidus was hypothesized until the diagnosis of Bartter syndrome type I was established by molecular evaluation. We conclude that in some patients with Bartter syndrome type I, hypokalemia and/or metabolic alkalosis may be absent in the first years of life and per- sistent metabolic acidosis or hypernatremia and hyper- chloremia may also be present. Molecular evaluation can definitely establish the diagnosis of atypical cases of this complex hereditary tubular disorder, which, in our expe- rience, may exhibit phenotypic variability.
Since IGF-I is an important chondrocyte growth factor, we sought to examine the intracellular mechanisms by which it exerts two of its pivotal effects, stimulation of proliferation and differentiation. We used the mesenchymal chondrogenic cell line RCJ3.1C5.18, which progresses spontaneously to differentiated growth plate chondrocytes. This differentiation process could be enhanced by exogenous IGF-I. Pharmacological inhibition of the phosphatidylinositol-3 (PI-3) kinase by LY294002, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)1/2 by U0126, the protein kinase (PK) A pathway by H-89 or KT5720, and the PKC pathway by bisindolylmaleimide suppressed IGF-I-stimulated cell proliferation. In contrast, IGF-I-enhanced early cell differentiation, as assessed by collagen type II and aggrecan gene expression, was not affected by MAPK/ERK1/2 pathway inhibition, but significantly diminished by inhibition of the PI-3 kinase, the PKC and the PKA pathway. Moreover, terminal differentiation of chondrocytes in response to IGF-I, as assessed by gene expression of alkaline phosphatase, Indian hedgehog, and collagen type X, were only interrupted by PI-3 kinase pathway inhibition. In conclusion, IGF-I exerts its differential effect on chondrocyte proliferation vs differentiation through the use of at least four partially interacting intracellular signaling pathways, whose activity is temporarily regulated. When chondrocytes progress from proliferating cells to early and terminal differentiating cells, they progressively inactivate IGF-I-related intracellular signaling pathways. This mechanism might be essential for the complex and cell stage-specific anabolic action of IGF-I in the growth plate.
The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators.Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins.Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis.Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system.Production of rRNA is abnormally elevated in Rb ؊/؊ p130 ؊/؊ fibroblasts.Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo.This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF.The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.
The growth plate is an important target tissue for insulin-like growth factors (IGFs), but little is known about the regulation of the IGF system during the developmental sequence of chondrocytes. We therefore examined the expression profile of IGF system components in proliferating vs. differentiating growth plate chondrocytes by use of two cell culture models of the growth cartilage. In rat growth plate chondrocytes in primary culture, IGF-I expression increased twofold during the process of differentiation. IGF-binding protein-3 (IGFBP-3) expression showed a biphasic pattern of with a twofold increase at the onset of differentiation and a downregulation in late differentiating chondrocytes to 25% of baseline levels; the expression patterns of IGFBP-2, -4 and -6 were not dependent on the developmental stage. In IGF- and IGFBP-3-deficient RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic cells, IGFBP-2 and -6 synthesis declined by 50% during differentiation. IGFBP-5 expression was markedly upregulated during the process of differentiation in both cell culture models. Although IGFBP-5 overexpression did not have an IGF-independent effect on RCJ cell differentiation, it promoted IGF-I-enhanced differentiation of these cells. A potential mechanism for this effect is the specific increase of Akt phosphorylation in IGFBP-5-overexpressing cells in the presence of IGF-I, indicating an increased activity of the phosphatidylinositol (PI) 3-kinase pathway. These data suggest that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression and imply a functional role for IGFBP-5 for upregulating IGF action during chondrocyte differentiation in vivo.
The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2167–279, IGFBP-2167–289, and IGFBP-2104–289 exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nm) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2104–289 for the stimulation of cell proliferation was 10 nm. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.
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