Regulation of mating in the budding yeast Saccharomyces cerevisiae by the zinc cluster proteins Sut1 and Sut2

The zinc cluster proteins Sut1 and Sut2 play a role in sterol uptake and filamentous growth in the budding yeast Saccharomyces cerevisiae. In this study, we show that they are also involved in mating. Cells that lack both SUT1 and SUT2 were defective in mating. The expression of the genes NCE102 and PRR2 was increased in the sut1 sut2 double deletion mutant suggesting that Sut1 and Sut2 both repress the expression of NCE102 and PRR2. Consistent with these data, overexpression of either SUT1 or SUT2 led to lower expression of NCE102 and PRR2. Furthermore, expression levels of NCE102, PRR2 and RHO5, another target gene of Sut1 and Sut2, decreased in response to pheromone. Prr2 has been identified as a mating inhibitor before. Here we show that overexpression of NCE102 and RHO5 also reduced mating. Our results suggest that Sut1 and Sut2 positively regulate mating by repressing the expression of the mating inhibitors NCE102, PRR2 and RHO5 in response to pheromone.     

An analysis of the Impact of <Emphasis Type="Italic">NRG1</Emphasis> Overexpression on the <Emphasis Type="Italic">Candida albicans</Emphasis> Response to Specific Environmental Stimuli

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.     

IGFBP3 deposited in the human umbilical cord mesenchymal stem cell‐secreted extracellular matrix promotes bone formation

The extracellular matrix (ECM) contains rich biological cues for cell recruitment, proliferationm, and even differentiation. The osteoinductive potential of scaffolds could be enhanced through human bone marrow mesenchymal stem cell (hBMSC) directly depositing ECM on surface of scaffolds. However, the role and mechanism of human umbilical cord mesenchymal stem cells (hUCMSC)‐secreted ECM in bone formation remain unknown. We tested the osteoinductive properties of a hUCMSC‐secreted ECM construct (hUCMSC‐ECM) in a large femur defect of a severe combined immunodeficiency (SCID) mouse model. The hUCMSC‐ECM improved the colonization of endogenous MSCs and bone regeneration, similar to the hUCMSC‐seeded scaffold and superior to the scaffold substrate. Besides, the hUCMSC‐ECM enhanced the promigratory molecular expressions of the homing cells, including CCR2 and TβRI. Furthermore, the hUCMSC‐ECM increased the number of migrated MSCs by nearly 3.3 ± 0.1‐fold, relative to the scaffold substrate. As the most abundant cytokine deposited in the hUCMSC‐ECM, insulin‐like growth factor binding protein 3 (IGFBP3) promoted hBMSC migration in the TβRI/II‐ and CCR2‐dependent mechanisms. The hUCMSC‐ECM integrating shRNA‐mediated silencing of Igfbp3 that down‐regulated IGFBP3 expression by approximately 60%, reduced the number of migrated hBMSCs by 47%. In vivo, the hUCMSC‐ECM recruited 10‐fold more endogenous MSCs to initiate bone formation compared to the scaffold substrate. The knock‐down of Igfbp3 in the hUCMSC‐ECM inhibited nearly 60% of MSC homing and bone regeneration capacity. This research demonstrates that IGFBP3 is an important MSC homing molecule and the therapeutic potential of hUCMSC‐ECM in bone regeneration is enhanced by improving MSC homing in an IGFBP3‐dependent mechanism.