Six decades of dentinogenesis research. Historical and prospective views on phosphophoryn and dentin sialoprotein.

The molecular basis underlying the mineralization process associated with the conversion of predentin to dentin is poorly understood. What is clear is that a unique set of non-collagenous proteins (NCPs) participate in the nucleation process and in hydroxyapatite growth during dentin formation. Phosphophoryn (PP), the most abundant NCP in dentin, is secreted by odontoblasts and appears at the mineralization front. Dentin sialoprotein (DSP), another NCP, also appears at the mineralization front, but only accounts for 5-8% of the weight of dentin NCPs. Functionally, PP is believed to be directly involved in tile nucleation process by virtue of its ability to bind to collagen type I, and its high affinity for calcium ions. Based on the analysis of the putative rat PP amino acid sequence, this latter activity is believed due to the highly phosphorylated character of PP, which results from the dual actions of casein kinases I and II at selected domains within PP. The precise role of DSP is currently unknown. In situ studies demonstrate that DSP is substantially expressed in odontoblasts and transiently expressed in preameloblasts. However, no information is currently available to directly explain DSP's role in mineralization. Genetically, we and others have now identified a novel DSP-PP bicistronic mammalian transcriptional unit, suggesting that the functional roles of these two NCPs may also be tightly coupled with respect to dentinogenesis. Certainly, further exciting studies are now needed to explain how this DSP-PP transcriptional unit is finally expressed: whether DSP and PP associate with one another, or with collagen at the mineralization front: and how selective mutations in either gene may influence dentin mineralization.

Interleukin 6 stimulates hepatic glucose release from prelabeled glycogen pools.

Cytokines, derived from a wide variety of cell types, are now believed to initiate many of the physiological responses accompanying the inflammatory phase that follows either Gram-negative septicemia or thermal injury. Because hypoglycemia (after endotoxic challenge) and hyperglycemia (after thermal injury) represent well-characterized responses to these injuries, we sought to determine whether hepatic glycogen metabolism could be altered by specific cytokines. Cultured adult rat hepatocytes were prelabeled with [14C]glucose for 24 h, a procedure that resulted in the labeling of hepatic glycogen pools that subsequently could be depleted (with concomitant [14C]glucose release) by either glucagon or norepinephrine. After the addition of a highly concentrated human monocyte-conditioned medium (MCM) or various cytokines to these prelabeled cells, [14C]glucose release was stimulated by MCM and recombinant human interleukin 6 (IL-6) but was not stimulated by other cytokines tested. Furthermore, only antisera to IL-6 were capable of reducing the glucose-releasing factor activity found in MCM. These data therefore suggest a novel glucoregulatory role for IL-6.

Microtubule depolymerization inhibits the regulation of alpha 1-acid glycoprotein mRNA by hepatocyte stimulating factor.

Hepatocyte stimulating factor (HSF, a polypeptide cytokine) is a major regulatory hormone responsible for hepatic acute-phase reactant (APR) induction following acute systemic injury. The mechanisms by which HSF regulates APR synthesis in the liver are unknown. Microtubules are involved in a number of polypeptide hormone-mediated events which can be modified, either positively or negatively, by microtubule depolymerizing agents. In this study we have used colchicine (a microtubule depolymerizing drug) to assess whether or not HSF-mediated changes in rat hepatic alpha 1-acid glycoprotein (AGP) or albumin mRNA levels require an intact microtubule cytoskeletal system. Cultured rat hepatocytes were pretreated for 30 min with either colchicine (10(-6) M), or the inactive isomer lumicolchicine (10(-6) M), or fresh medium. Following pretreatment, purified murine macrophage HSF (10 units/ml) was added and the cells were incubated for an additional 12 h. Colchicine, but not lumicolchicine, significantly inhibited the HSF-dependent regulation of mRNA for the positive APR, AGP, but had no effect on the mRNA levels of albumin, a negative APR. Furthermore, removal of colchicine from previously inhibited cultures allowed HSF to restimulate AGP mRNA expression. These data suggest that microtubules may play a regulatory role in controlling the expression of the genes for positive acute-phase proteins and may explain the temporal differences found in vivo between positive and negative APR expression.

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures.

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.

Evidence that in vitro adherence of Klebsiella pneumoniae to ciliated hamster tracheal cells is mediated by type 1 fimbriae.

Clinical isolates of fimbriated and nonfimbriated Klebsiella pneumoniae were examined for the ability to adhere to hamster tracheal cells cultured in vitro. Fimbriated-phase K. pneumoniae adhered preferentially to ciliated cells, whereas nonfimbriated-phase organisms were not adherent. The adherence was inhibited by D-mannose but not D-glucose, suggesting that type 1 fimbriae serve as the adhesin in the attachment process.

Restoration of the LPS responsive phenotype in C3H/HeJ macrophage hybrids: LPS regulation of hepatocyte-stimulating factor production.

The fusion of thioglycollate-elicited peritoneal macrophages from lipopolysaccharide (LPS) non-responsive C3H/HeJ mice to an HPRT-negative variant of the murine macrophage cell line P388D1 has resulted in the derivation of a series of macrophage hybrids. Following exposure to LPS, these hybrids now produce the cytokine hepatocyte-stimulating factor (HSF) which induces the synthesis of the acute-phase reactant alpha 2-macroglobulin in primary rat hepatocyte cultures. The concentration of extracellular HSF was dependent upon both the duration and amount of LPS, with optimal HSF being detected after 72 hr incubation with 10 micrograms/ml of LPS. Parallel LPS-stimulated cultures treated with 10(-6)M dexamethasone did not secrete detectable amounts of HSF. Both the molecular weight (29,000 MW), and the fact that HSF activity was not inhibited by an antiserum directed against murine interleukin-1 alpha (IL-1 alpha), suggests that HSF and IL-1 are distinct cytokines. Therefore, macrophage hybrids have been derived which have acquired the LPS-responsive phenotype and which synthesize the cytokine HSF following LPS stimulation. This phenotype appears stable since similar results have been observed with these hybrids after in vitro culture for over 8 months.

An in vitro system for the study of tracheal epithelial cells.

Enzymatically dissociated hamster tracheal epithelial (HTE) cells were cultured on collagen coated Millicell filters. Within 3-5 days after being placed in culture large numbers of ciliated and mucus cells began to appear. By 1 week the HTE cells closely resembled those seen in vivo, i.e. columnar morphology and organelle polarity. After 4 weeks in vitro the HTE cultures were still able to maintain the polarity and overall columnar morphology found in in vivo tissue. There was, in addition, no apparent degradation of the collagen substrate. Auto-radiographic data indicated that there was an initial period of high DNA synthesis during the first 3 days in vitro. This was followed by a second phase in which, by day 6, the amount of DNA synthesis was greatly reduced. Analysis of the numbers of ciliated cells relative to non-ciliated cells demonstrated that between days 5 and 8 there was an increase in the percentage of ciliated cells, suggesting that cellular differentiation (i.e. ciliogenesis) follows cellular proliferation. The results of this study show that when HTE cells are grown on collagen-coated Millicell filters there is a significant improvement in cell growth and morphology yielding cells that are very similar to those present under in vivo conditions. Moreover, since there is no degradation of the collagen substrate, HTE cultures may be suitable for long-term studies of respiratory tract epithelia.

Regulation of fibrinogen synthesis by plasmin-derived fragments of fibrinogen and fibrin: an indirect feedback pathway.

The effect of plasmin-derived fibrinogen fragments on the biosynthesis of fibrinogen was investigated in cultured monolayers of rat hepatocytes. Incubating the cells with several concentrations of either fibrinogen or fibrin fragment D or E had no effect on the synthesis and secretion of fibrinogen by these cells. However, if the fragments were incubated with isolated peripheral blood leukocytes, they caused these cells to secrete a factor that when added to the hepatocytes caused an increase in fibrinogen synthesis 4- to 6-fold over controls. Moreover, the hepatocyte-stimulating factor also affected the production of several other proteins produced by the hepatocyte. These results demonstrate that both fragments D and E can stimulate hepatic fibrinogen synthesis via an indirect leukocyte-mediated pathway.

An in vitro bioassay for leukocytic endogenous mediator(s) using cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes were used to assay for the presence of leukocytic mediator(s) (LEM), a neutrophil derived protein(s) capable of stimulating the synthesis of acute-phase plasma proteins when injected into rats. In the presence of physiological concentrations of dexamethasone (40 mM), the hepatocytes secreted a variety of plasma proteins as demonstrated by crossed immunoelectrophoresis. The addition of LEM to hepatocytes increased the secretion of several acute-phase related plasma proteins, including fibrinogen and hepatoglobin, and decreased albumin secretion. These results mimic the acute-phase response observed in the intact animal. Fibrinogen secretion was used as a quantitative marker for determining LEM activity. The rate of fibrinogen secretion depended upon both the concentration of dexamethasone and LEM present during a given 24-h assay period. One unit of LEM activity is defined as that concentration of LEM capable of producing a 50% maximal stimulation of fibrinogen secretion.

Cystinotic fibroblasts are depleted of free-cystine by acid pH medium.

The free (nonprotein) cystine content of human cystinotic fibroblasts was found to vary with the pH of their culture medium. Intracellular cystine content was highest at alkaline medium pH's and lowest at acidic medium pH's. When maintained in medium of pH 6.3, cystinotic fibroblasts lost 50% of their free-cystine content in 6 hr and over 95% in 30 hr. Cystine was the only amino acid to show significant variation with changes in the pH of the culture medium.

Cystinotic fibroblasts accumulate cystine from intracellular protein degradation.

Fibroblasts derived from patients with cystinosis, an autosomal recessive condition, accumulate the disulfide amino acid cystine within lysosomes. The metabolic defect leading to the cystine accumulation and the source from which the cystine is derived are unknown. In this report we present data showing that cystine in these cells accumulates from the degradation of endogenous protein. This conclusion is based upon: (i) no demonstrable synthesis of cystine from serine; (ii) no difference in cystine reaccumulation between glutathione-depleted and non-glutathione-depleted cystinotic cells; (iii) recovery of labeled cystine only when the protein pool is labeled; (iv) reversible inhibition of cystine reaccumulation by known inhibitors of lysosomal protein degradation (chloroquine and NH4Cl).