Wound healing and the dysregulated events leading to fibrosis both involve the proliferation and differentiation of fibroblasts and the deposition of extracellular matrix. Whether these fibroblasts are locally derived or from a circulating precursor population is unclear. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that enter sites of tissue injury to promote angiogenesis and wound healing. We have found that CD14+ peripheral blood monocytes cultured in the absence of serum or plasma differentiate into fibrocytes within 72 h. We purified the factor in serum and plasma that prevents the rapid appearance of fibrocytes, and identified it as serum amyloid P (SAP). Purified SAP inhibits fibrocyte differentiation at levels similar to those found in plasma, while depleting SAP reduces the ability of plasma to inhibit fibrocyte differentiation. Compared with sera from healthy individuals and patients with rheumatoid arthritis, sera from patients with scleroderma and mixed connective tissue disease, two systemic fibrotic diseases, were less able to inhibit fibrocyte differentiation in vitro and had correspondingly lower serum levels of SAP. These results suggest that low levels of SAP may thus augment pathological processes leading to fibrosis. These data also suggest mechanisms to inhibit fibrosis in chronic inflammatory conditions, or conversely to promote wound healing.

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes, which are associated with and are at least partially responsible for wound healing and fibrosis in multiple organ systems. Signals regulating fibrocyte differentiation are poorly understood. In this study, we find that when added to human PBMCs cultured in serum-free medium, the profibrotic cytokines IL-4 and IL-13 promote fibrocyte differentiation without inducing fibrocyte or fibrocyte precursor proliferation. We also find that the potent, antifibrotic cytokines IFN-gamma and IL-12 inhibit fibrocyte differentiation. In our culture system, IL-1beta, IL-3, IL-6, IL-7, IL-16, GM-CSF, M-CSF, fetal liver tyrosine kinase 3, insulin growth factor 1, vascular endothelial growth factor, and TNF-alpha had no significant effect on fibrocyte differentiation. IL-4, IL-13, and IFN-gamma act directly on monocytes to regulate fibrocyte differentiation, and IL-12 acts indirectly, possibly through CD16-positive NK cells. We previously identified the plasma protein serum amyloid P (SAP) as a potent inhibitor of fibrocyte differentiation. When added together, the fibrocyte-inhibitory activity of SAP dominates the profibrocyte activities of IL-4 and IL-13. The profibrocyte activities of IL-4 and IL-13 and the fibrocyte-inhibitory activities of IFN-gamma and IL-12 counteract each other in a concentration-dependent manner. These results indicate that the complex mix of cytokines and plasma proteins present in inflammatory lesions, wounds, and fibrosis will influence fibrocyte differentiation.

Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that inhibits the proliferation of wild-type and aprA- cells;this activity is not secreted by aprA- cells. AprA purified by immunoprecipitation also slows the proliferation of wild-type and aprA- cells. Compared with wild type, there is a higher percentage of multinucleate cells in the aprA- population,and when starved, aprA- cells form abnormal structures that contain fewer spores. AprA may thus decrease the number of multinucleate cells and increase spore production. Together, the data suggest that AprA functions as part of a Dictyostelium chalone.

Developing Dictyostelium cells form large aggregation streams that break up into groups of 0.2 × 10 5 to 1 × 10 5 cells. Each group then becomes a fruiting body. smlA cells oversecrete an unknown factor that causes aggregation streams to break up into groups of ∼5 × 10 3 cells and thus form very small fruiting bodies. We have purified the counting factor and find that it behaves as a complex of polypeptides with an effective molecular mass of 450 kD. One of the polypeptides is a 40-kD hydrophilic protein we have named countin. In transformants with a disrupted countin gene, there is no detectable secretion of counting factor, and the aggregation streams do not break up, resulting in huge (up to 2 × 10 5 cell) fruiting bodies.

No abstract

High-density microarrays were used to profile circadian gene expression in Neurospora crassa cultures grown in constant darkness. We identified 145 clock-controlled genes (ccgs). The ccgs peaked in mRNA accumulation at all phases of the day, with the majority peaking in the late night to early morning. The predicted or known functions of the ccgs demonstrate that the clock contributes to a wide range of cellular processes, including cell signaling, development, metabolism, and stress responses. Although the period of rhythm of most of the ccgs was found to depend on the well characterized FREQUENCY (FRQ)-based oscillator, three ccgs appeared to have a rhythm that was significantly short in the long period (29-h) frq 7 mutant strain. These ccgs accumulate mRNA rhythmically with a circadian period in a frq-null strain, confirming the existence of a second oscillator in N. crassa.clock-controlled gene ͉ microarray ͉ frequency C ircadian rhythms are endogenous self-sustaining oscillations that are regulated by a central pacemaker composed of one or more biochemical oscillators (1, 2). These rhythms are observed in a wide variety of organisms, ranging from daily rhythms in photosynthesis in cyanobacteria and plants to activity and sleep-wake cycles in rodents and humans. An important aspect of rhythmicity involves control of specific target genes by oscillators; however, this remains one of the least understood areas in chronobiology.In Neurospora crassa, the well characterized FREQUENCY (FRQ)-based oscillator consists of an interlocked autoregulatory molecular feedback loop containing positive PAS domaincontaining elements WHITE COLLAR-1 (WC-1) and -2 and a negative element (FRQ), which depresses the activity of the positive elements (3). WC-1 is a blue-light photoreceptor that links the circadian oscillator to the external environment (4, 5). Using subtractive hybridization and differential screens of timeof-day-specific libraries, eight clock-controlled genes (ccgs) have been identified in N. crassa (3). All of the ccgs peak in expression in the late night͞early morning, and the associated proteins function in intermediary metabolism, stress responses, and development. Mutation of the frq gene affects the rhythmic expression of all of the known ccgs, suggesting that they are controlled by the FRQ-based oscillator. However, circumstantial evidence suggests that the N. crassa clock is built by using more than one oscillator (6-8), and these other FRQ-less oscillators (FLOs) may participate in the regulation of ccgs.In this study, we used microarray technology to search for ccgs in N. crassa. Our data reveal the importance of the clock in the life of the fungus and provide molecular evidence for the existence of the FLO. (9) but does not affect the clock itself. Growth media (Vogel's and Fries' minimal media), vegetative growth conditions, and crossing protocols are described (10). Materials and MethodsCulture Harvesting Conditions. For rhythmic RNA analyses, the clock was synchronized by a light-to-dark transition ...

Using an antisense construct of the discoidin gene transfected into Dictyostelium, we have repressed the expression of the three endogenous discoidin genes. Transformants exhibit a greater than 90% reduction in accumulated discoidin mRNA and protein. Nuclear run-on assays show that both the endogenous and the antisense genes are transcribed. Since only minor amounts of endogenous gene transcripts and none from the antisense gene can be detected on blots, we suggest that hybrids are formed within the nucleus and are rapidly degraded. Discoidin is believed to play a role in cell-substratum interaction and exhibits homologies to fibronectin. Discoidin-minus mutants exhibit the developmental phenotype of not streaming on a plastic surface. Antisense transformants show a similar phenotype and are thus phenocopies of these mutants.

Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD– cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a ∼150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.